Review of Literature

REVIEW OF LITERATURE

From times immemorial, tuberculosis has ranked amongst the most feared and dreaded diseases that have afflicted mankind. Persuasive biological and historical evidence support the speculation, that M tuberculosis evolved into a

human infective pathogen from M bovis sometime after the domestication of cattle

between 8000 and 4000 B.C. To the extent that diagnosis can be made accurately,

this coincides well with the first good archeological and paleopathological indications of spinal tuberculosis, believed to be caused by M bovis. It has been only recently that Salo et al (1994), using DNA amplification techniques on

samples from a 1000 year old mummified remains of an adult female found in

southern Peru, demonstrated the existence of a 'tuberculosis complex' bacteria which they identified as M tuberculosis.

The origin and spread of tuberculosis in human population remains

shrouded in mystery. However, it is believed to be closely associated with the

migration of the pastoralist cattle herders of the Indo-European tribes that moved

into the Indus valley around 1500 B.C. This initially resulted in conditions such as overcrowding, malnutrition and war, which favored the spread of infectious

diseases such as tuberculosis (Dubos and Dubos, 1952). It has been hypothesized that the disease must have started in a milk drinking population of Indo-European

lineage, due to the consumption of infected milk, and later spread to other areas along with Aryan invasions. The earliest tangible mentions of the symptoms of pulmonary tuberculosis are found in clay tablet inscriptions of the Assyrian king

Assurbanipal (668-626 B.C) and theAtharva Veda (400 B.C).

Although by about 1000 A.D, Avicenna was advocating the cavitary and

inflammatory nature of tuberculosis, the tubercle itself was fmally identified and

understood only with the work of Franciscus Sylvius. Important contributions to

the association between tubercle formation and the development of tuberculosis were also made by the work of Bayle and Laennec (Flick, 1925). The turning point in the history of tuberculosis occured in 1882, when Robert Koch described the

isolation of the causative organism of this dreaded disease. Koch's studies also led to the development of the Old Tuberculin and the description of the necrotic hypersensitivity reaction (Koch, 1891). Old tuberculin and the reaction it elicited were then used by Clemens Von Pirquet in 1907 for the development of the

tuberculin test. Though, it is to date the most widely used test for tuberculosis, unfortunately, it has limitations both due to its lack of specificity and inability to

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distinguish between active disease, or cross sensitization to other related

mycobacteria. The principles of vaccination were established by Pasteur, and many

workers attempted to attenuate the tubercle bacillus for use as a vaccine. A vaccine

was eventually developed by Calmette and Guerin after passaging a bovine tubercle bacillus for 230 subcultures on a glycerinated beef-bile-potato medium (Calmette et al, 1927). Though, BCG remains the most widely used vaccine and

has been administered to over 3 billion people since 1948, its protective efficacy (Fine, 1989) and safety, especially in immunocompromised individuals (Braun and

Cauthen, 1992) is questionable. Various strategies are therefore underway to develop more effective recombinant BCG and subunit vaccines (Stover et al, 1991;

Barletta et al, 1990; Anderson, 1994; Horwitz et al, 1995)

THE CAUSATIVE ORGANISM

Tuberculosis is caused by the organism Mycobacterium tuberculosis

belonging to the genera Mycobacterium which contains more than 54 recognized

species (Wayne and Kubica, 1986). The presence of mycolic acids is a

characteristic taxonomic feature of mycobacteria and is responsible for the property of 'acid fastness'. M tuberculosis may be recognized by the development

of rough, eugenic colonies with a characteristic buff tint after 2-4 weeks of culture.

The bacilli reduce nitrate, produce niacin, lose catalase activity after heating at 68°

C, and are usually sensitive to streptomycin, p-amino salicylic acid and isoniazid,

unless the isolate is drug resistant. Both M tuberculosis H37Rv and H37Ra were originally derived froin the same parental strain (H 3 7) by separation on the basis

of subtle differences in colony morphology and their virulence for rabbits and

guinea pigs (Steenken and Gardner, 1946). Apart from these biochemical tests,

more sophisticated and rapid tests are now available for identifying and distinguishing M tuberculosis from the other bacteria of the M tuberculosis

complex. Primary among them are tests based on DNA probes (Evans et al, 1992),

HPLC (Thibert and Lapierre, 1993), PCR-RFLP (Plikaytis et al, 1992) and 16S rRNA/sequencing (Kirschner et al, 1993).

It is the envelope which essentially distinguishes species of the Mycobacterium genus from other prokaryotes. Mycobacteria lack the classical endotoxin LPS associated with gram -ve bacteria and are phylogenetically closer to gram +ve bacteria (Draper, 1982), and yet, share some features of the gram -ve bacteria such as the presence of diaminopimelic acid. Thus, mycobacterial cell wall exhibits some properties common to both gram +ve and gram -ve bacteria but

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is characterized by the presence of features that are uniquely its own (Me Neil et

a/, 1991 ). The most distinctive among these is the presence of Mycolic acid that

constitutes more than 50% by weight of the cell wall envelope. The cell wall core

is composed of three covalently attached macromolecules - peptidoglycan, arabinoglycan and mycolic acids. Mycolic acids are anchored to the arabinan chains of the arabinogalactan. Various other glycolipids, polysaccharides and

proteins are associated with the cell wall to form the cell envelope. Prominent

among these is the lipopolysaccharide lipoglycan called lipoarabinomannan

(LAM). Studies carried out by various groups have shown LAM to cause

nonspecific suppression ofT cell activation (Kaplan et a/, 1987), inhibition of

macrophage activation (Sibley et a/, 1988) and inhibition of antigen responsiveness of human PBMCs (Moreno et a/, 1988). Other prominent constituents of the cell wall are the trehalose based glycolipids, glycopeptidolipids and phenolic glycolipids (Brennan, 1988).

TRANSMISSION

Understanding the transmission of tuberculosis began with Robert Koch's

description of M tuberculosis in 1882. In humans, tuberculosis results from an infection with one of the three organisms that comprise the M tuberculosis complex : M tuberculosis, the human tubercle bacillus, M bovis, the bovine tubercle bacillus; or M africanum, a mycobacterium with biochemical features common with M tuberculosis and M bovis (Grange and Yates, 1989). Koch

demonstrated the aerosol transmission of M tuberculosis to guinea pigs in 1884,

and identified human sputum as the most important vehicle for the dissemination

of the bacteria.

Central to the concept of airborne transmission is the droplet nucleus.

Coughing, one of the main symptoms of tuberculosis, produces droplets which are

one to five microns in size, containing one to three bacteria surrounded by a layer

of moisture. These droplets remain suspended in the air and when breathed in, they become deposited in the respiratory airway. A few of them manage to cross the mucosal barrier and form focus of infection in the lung bed. Studies carried out by Riley and coworkers between 1957 - 1962 established the mechanism of aerosol transmission from infected patients to guinea pigs, by placing guinea pigs in the ventilation ducts of the TB unit in a hospital. Riley also showed that effective chemotherapy for TB prevented aerosol transmission (Riley, 1962). Factors that determine the likelihood of transmission of infection, include the number, viability or virUlence of orgarusms, environmental factors such as

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ventilation, humidity or exposure to ultraviolet light and the susceptibility of the

rece1ver.

INVASION AND PHAGOCYTOSIS

The epidemiological fate of M tuberculosis is linked to its intracellular

survival. After M tuberculosis infects a host, it may be immediately eliminated

and hence lead to no infection or it may lie dormant inside the host indefmitely

and lead to a state of latent infection. It may also cause a disease soon after

infection (primary tuberculosis) or years later (reactivation tuberculosis). Phagocytosis of M tuberculosis by mononuclear cells is the frrst event in the complex host pathogen interaction that may lead either to latent infection or active

disease. Work carried out by Schlesinger and coworkers in 1990, showed that

phagocytosis of M tuberculosis by human monocyte derived macrophages is

mediated by complement receptores CRl and CR3 after opsonization by C3

component of the complement. Further studies by the same worker (Schlesinger,

1993) established the involvement of mannose receptors in the phagocytosis of virulent but not avirulent strains of M tuberculosis. He suggested that differential

use of pathways of entry may influence the survival of virulent or avirulent strains, inside the macrophage. In 1957, Shepard reported that M tuberculosis can enter

the non phagocytic HeLa cells. This observation brought forth the fact that apart from macrophage surface and serum factor mediated phagocytosis, products of

mycobacteria itself may be important in directing its entry into cells. In 1993,

Arruda and colleagues identified a 1535 base DNA fragment of M tuberculosis

that could confer a property of invasiveness on a non pathogen strain of E. coli.

Since then studies carried out by McDonough et a/ (1995) and Bermudez and coworker (1996) have established that virulent M tuberculosis can successfully invade and replicate inside epithelial cells. This has important implications for hematogenous spread of infection. Post phagocytosis, the bacteria become

enclosed in membrane bound vesicles or phagosomes that susequently fuse with lysosomes. They may be then digested by lysosomal enzymes which are released

in the phagosome-lysosomal compartment. Mycobactenum are equipped with a number of mechanisms which help it in avoiding macrophage mediated killing. Sulfatides of M tuberculosis have been proposed to be involved in inhibiting the phagolysosome fusion (Goren et a/, 1976). Sturgill - Kosycki and colleagues in 1994, demonstrated that M avium could selectively exclude the proton- ATPase pump from the phagosome, thereby avoiding acidification of the phagolysosome.

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Lipoarabinomannan, a prominent constituent of mycobacterial cell wall has been shown to inhibit macrophage activation (Sibley et al, 1988). Another mechanism

by which pathogens avoid the antimicrobial effects of phagolysosomes is by

escaping out into the cytoplasm. McDonough and coworker (1993) observed that

phagosomes containing virulent (H37Rv) but not avirulent bacteria (BCG/H37Ra) did not fuse efficiently with secondary lysosomes. Further, the virulent bacteria evaded the microbicidal environment of the macrophage by escaping from the fused phagolysosome into nonfused vesicles or the cytoplasm.

Mononuclear phagocytes generate reactive oxygen intermediates such as

superoxides, hydrogen peroxide and hydroxyl radicals, which may have

microbicidal effects (Murray et al, 1979; Nathan et al, 1983). Although, there is

evidence that these oxygen intermediates contribute to mycobacterial killing, their

role in killing the phagocytosed tubercle bacilli remains unestablished.

Mycobacteria are equipped with enzymes such as superoxide dismutase and catalase that can interfere with the generation of Reactive Oxygen Intermadiates (ROis ), and may contribute to intracellular survival of the bacteria. Although, there is evidence that some catalase deficient strains of M tuberculosis are more

susceptible to hydrogen peroxide, such a feature is not associated with H37Ra (Jackett et al, 1978, 1980). Using the murine macrophage cell line D9, which is

known to be dificient in ROI generation, Chan and colleagues (1992) showed that activated macrophage mediated killing of M tuberculosis was independent of ROI generation.

Reactive Nitrogen Intermediates (RNis) are generated from the metabolic

pathway that begins with the oxidation of L-arginine by the action of the enzyme nitric oxide synthase. RNis regulate a number of biological functions such as

vasodialation (Stefano et al, 1995), neurotransmission (Belvisi et al, 1992) and

lymphocyte proliferation. Murine macrophages express an inducible form of the enzyme nitric oxide synthase upon stimulation with IFNy or LPS and upon infection with BCG (Stuehr and Marietta, 1987). The observations of Flesch and Kaufmann (1991) suggested that the intracellular growth of mycobacteria is

inhibited by factors other than oxygen intermediates. Later the same workers as well as Denis ( 1991) demonstrated that RNis derived from the inducible pathway were the main mediators of intracellular mycobacterial killing. There exists a strain to strain variation in the susceptibility of mycobacteria to RNis and such differences may manifest in the wide spectrum of host infection and disease observed after M tuberculosis infection. The role of RNis generated by human macrophages in antimicrobial mechanisms remained controversial till recently

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(Schneemann et al, 1993), as human cells do not respond to the same stimuli as

murine macrophages .. It is likely that this reflects a difference in the signal

transduction pathways operating· in the two kinds of cells. Recent reports have

demonstrated the detection of RNis and iNOS activity in human macrophages, including those in monocyte derived macrophages (Pietraforte et ai, 1994), bronchial epithelial cells in asthmatic. patients (Hamid et al, 1993) and ·alveolar macrophages in lungs with chronic inflammation (Kobzik et al, 1993).

Nitric oxide, in addition to its role as a cellular messenger at low

concentrations, has been shown to play a decisive role in inflammatory and

autoimmune tissue injury because of its immunoregulatory properties when synthesized at high concentrations (Nussler and Biliar, 1993; Becherel et a!,

1994). Nitric oxide (NO} has many actions that are appropriate for a proinflammatory agent. It is made by numerous cell types at sites of inflammation and it increases blood flow and vascular permeability. NO has cell and tissue destructive abilities; it can also induce cyclooxygenase, cause pain, destroy certain

protease inhibitors and enhance IL-l, TNF and NADPH oxidase activities in

myeloid cells (Magrinat et al, 1992; Clancy and Abramson, 1995). NO production

may be augmented by several substances, including cytokines, growth factors, immune complexes and bacterial products. Since superoxide may convert NO to

peroxynitrite (a potent pro inflammatory molecule), concomitant production of NO

and superoxide may further amplify the inflammatory state (Privalle et al, 1996).

Enhanced production of nitric oxide by endothelial cells leads to vasodialation, cause inhibition of VCAM-1 expression and has been reported to inhibit adhesion

of platelets and neutrophils to the endothelium, but a similar study on the effect of

NO on macrophages is not available. The function of all forms of nitric oxide

synthase enzymes can be inhibited by N- substituted arginine analogs such as NG­

monomethyl L-arginine (NMMA), aminoguanidine or N-nitroarginine methylester. The availability of such inhibitors allows assessment of the effects of the arginine­

NO pathway in vitro and in vivo (Nozaki et al, 1997). Studies using NO are often

hampered by the fact that it is a short lived molecule with a half life of less that 10

sec. in vitro. Many workers have, therefore, made use of NO donors such as

sodium nitroprusside (SNP), and 3-morpholinosydnonimine (SIN-I) to study the biological effects of nitric oxide (Bauer et al, 1997).

PATHOGENESIS OF PRIMARY INFECTION

Only droplet nuclei containing one to three bacilli are able to reach the alveolar spaces where they may establish an infection depending on the virulence

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of the bacteria and the susceptibility of the host. Animal studies carried out by Lurie and Dannenberg have contributed in a major way in understanding the pathogenesis of primary TB infection. In 1989, Dannenberg described the various

stages of infection. In the first stage, alveolar macrophages ingest the tubercle

bacilli and transport them to regional lymph nodes. Infected macrophages release

chemotactic factors, such as complement component C5a, that attract additional macrophages and circulating monocytes to the site. Macrophages containing

dividing bacilli may die, releasing more bacilli and cellular debris that also attract monocytes.

The second stage, named the 'Symbiotic stage' occurs from day 7 to 21 (Lurie, 1964). The balance between microbial virulence and host defense results in

macrophages that are unable to completely eliminate the bacterial load. As a result,

the number of bacteria increase logarithmically. inside unactivated macrophages.

The third stage which occurs after 3 weeks is characterized by the onset of cell mediated immunity and delayed type hypersensitivity (DTH). Macrophages are activated by IFNy released from Th1 cells and attain the microbicidal ability to

restrict the growth of intracellular bacilli (Lurie 1964, Nardell 1993). In the

process, a number of infected macrophages also die, resulting in the formation of a

nodule or granuloma. Granulomas are organized collection of epitheloid cells and

giant cells which show evidence of activation and are surrounded by lymphocytes

and capillaries (Fig. A). Macrophages entering the tubercle can become highly activated, ingest and successfully destroy bacilli, or remain poorly activated, such that they ingest but fail to destroy the bacilli and contribute to the central

caseation. Some caseous foci become replaced by fibrous tissue and may lead to

the formation of a Ghon focus. Tubercle bacilli, either free or within macrophages,

drain along the regional peribronchial lymphatic channels to the tracheobro~chial

lymph nodes, evoking caseating granulomas at this site. These series of events constitute primary tuberculosis and the lesions may either heal or progress to a diseased state. Granulomas that portray the appearance of a healed lesion may still reactivate months or years later in response to factors that are still poorly characterized.

In the fourth stage, hydrolytic enzymes liquify caseum, transforming it into

a rich medium that can support the extracellular growth pf mycobacteria. Excessive DTH reactions may lead to the collapse of bronchial walls. Once bronchial structures are involved, the liquified material may be expectorated as droplet nuclei and lead to the spread of infection.

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Epithelioid cell

Fig. A. Schematic representation of a granuloma

Review of Literature

GENETIC FACTORS AND THE ROLE OF BeG GENE IN TUBERCULOSIS

Most circumstantial evidence supporting the role of genetic factors in mycobacterial resistance was derived from vaccination trials with live, attenuated

M bovis BCG (Lotte eta/, 1984). In several of the major vaccination trial studies

(Chaparas, 1982), the protective efficacy of BCG has been found to vary widely,

and it has been speculated that this variation may have arisen from genetic differences between the study populations. The first conclusive evidence that genetic resistance mechanisms were operative in susceptibility to infection with M

tuberculosis H37Rv, were provided by Lurie and Dannenberg who initiated a systematic analysis of the genetic component involved in the control of susceptibility to TB in rabbits (Lurie and Dannenberg, 1965). Genetic analysis of

BCG susceptibility im mice led to observations that were compatible with the

presence of a single, dominant acting BCG resistance locus termed Beg (Gros et

a/, 1981 ). Phenotypically, two alleles of the gene can be distinguished; a resistant

allele (Bcg7) and a suscepibility allele (BegS). Later, in addition to BCG, the Beg

gene was also shown to regulate host resistance in vivo to other mycobacteria such as M lepramurium (Brown et al, 1982), M avium (Appelberg eta/, 1990) as well

as non mycobacterial infections such as those caused by Salmonella and Leishmania (Plant eta/, 1982). One important aspect of the Beg gene phenotype is

that it strictly controls the early innate phase of mycobacterial growth in reticuloendothelial tissues. The fact that Beg gene regulated early, nonimmune resistance to a variety of intracellular pathogens implicated the macrophage as the

cell type expressing the gene. Several groups have performed in vivo cell depletion and cell transfer experiments to determine the cellular basis of the Beg gene.

Conclusive proof that macrophages expressed the Beg gene was provided by in

vitro experiments showing that Bcgr macrophages were capable of significantly

reducing the growth of BCG (Stach et a/, 1984), M intracellulare (Goto et al,

1989) as well as L. donovani (Croaker et a/, 1987). Macrophages isolated from

resistant mice demonstrated a greater magnitude of hexose monophosphate shunt and respiratory burst activity after phagocytosis of BCG (Denis et a/, 1988) or

upon stimulation with IFNy (Blackwell eta/, 1988) as compared to macrophages from susceptible strains. Other studies have established that macrophages from resistant animals consistently display greater expression of other activation markers such as class II Ia antigen (Zwilling et al, 1994), LPS elicited TNFa

production (Blackwell et a/, 1991) and support of antigen specific and non­specific T cell proliferation (Kaye eta/, 1989).

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IMMUNOLOGY OF TUBERCULOSIS

Mycobacteria, in common with other intracellular parasites owe their

virulence to their ability to survive within the macrophages. Protective immune

response in mycobacterial disease are of the cell mediated type and serve to

enhance the ability of the macrophage to inhibit or destroy the invaders. Humoral immune response, i.e., antibody production certainly does occur, but there is no

evidence that they play any major role in the host defense (Grange 1984). Persons with defective cell mediated immunity, such as those with human immunodeficiency virus infection and chronic renal failure, are at markedly

increased risk for tuberculosis, whereas persons with defective humoral immunity, such as those with sickle cell disease and multiple myeloma, show no increased

predisposition to tuberculosis. Most experimental evidences indicate that antimycobacterial immune defenses are mediated primarily by T lymphocytes and macrophages and adoptive transfer of resistance against tuberculosis in animal

models has been shown to be mediated by T cells (Orme et al, 1983).

CELL MEDIA TED IMMUNE RESPONSE

Macrophages of the lung are the first cells parasitized in human pulmonary

tuberculosis and therefore play a central role in the disease; how these cells

interact with the bacilli may be the key question for understanding resistance. In

addition to their role as host cells that either support or restrict the intracellular

growth of mycobacteria, mononuclear phagocytes also produce and release cytokines such as TNFa, IL-l, IL-6, IL-8 etc. which are central to the immunoregulation of the disease (Steele et a/, 1986; Schauf et a/, 1993). Sensitized CD4+ T cells are readily detectable in humans exposed to

mycobacteria, as evidenced by in vitro blastogenesis in response to mycobacterial

antigens and by tuberculin hypersensitivity in vivo (Molloy and Kaplan, 1996). T cell responsiveness correlates inversely with disease progression in terms of both

low blastogenic responses to mycobacterial antigens in vitro (Toosi et al, 1986)

and reduced or absent skin tuberculin hypersensitivity among patients with

advanced or uncontrolled disease (Krause, 1922). It is now recognized that a subset of activated CD4+ T cells that secrete the key antimicrobial cytokine IFNy are responsible for the initial expression of protective immunity to M tuberculosis

(Cooper eta/, 1993), resulting in cessation of bacterial growth and some degree of

bacterial clearance (Orme and Collins, 1994).

Although it is generally believed that ens+ cells may not play an important role in mycobacterial infection, results to the contrary have been provided by some

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workers. Kaufmann (1989) presented evidence that both CD4+ and CDS+ T cells are generated in experimental tuberculosis in mice and depletion of either T cell subset resulted in a significant increase of the bacterial load, with CD4 + T cell depleted mice showing more severe effects. Studies carried out by Flynn et al

(1992) and Orme (1993) support the contention that CDS+a~ -T cells are also

involved in protective immunity against M tuberculosis infection, especially so in the lungs. A role for yo T cells in immunity to tuberculosis is supported by several

lines of evidence, including their accumulation in infected tissue (Modlin et a/, 1989), their secretion of IL-2 and IFNy in response to mycobacterial antigens

(Tsukaguchi et al, 1995) and by data showing their recognition of non peptide low molecular weight antigens of the bacillus (Tanaka eta/, 1995). A recent study by Ladel and colleagues (1995) has shown increased bacterial growth in yo TCR gene

disrupted mice, supporting the contention that they play a role in protective

immunity. Although human monocytes chronically infected with BCG are not

recognised efficiently by CD4+ T cells, they are preferentially recognised and

lysed by IL-2 activated lymphokine activated killer (LAK) cells (Molloy et a/,

1993).1n vivo, the generation ofLAK cells following vaccination with viable BCG

has been documented in experimental animals, although the mechanism of target

cell recognition by antigen independent non MHC restricted LAK cells remain poorly understood (Young, 1989). Generation of LAK activity in vitro requires high level of IL-2. While it is impossible to directly measure concentrations in

vivo, lymphokines are readily detectable m granulomas using

immunohistochemical techniques; and the architecture of the site is such that any lymphokine produced there is likely to be sequestered and concentrated.

Therefore, the sites of mycobacterial infection may constitute an ideal

microenvironment for the generation ofLAK cells (Barnes eta/, 1993).

Secreted proteins are considered to have an important role in inducing

immunity to M tuberculosis infection. Mendez et a/ (1995) analysed the proliferation of PBMCs from tuberculin positive or negative healthy controls and tuberculosis patients in response to whole bacilli, 3 8 KDa secreted protein and the 65KDa, 71KDa and 10KDa heat shock proteins. Comparing the proliferative responses between these antigens , it was observed that the 1 OKDa antigen showed a strong T cell response which was comparable to that seen with whole bacilli.

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CYTOKINES IN TUBERCULOSIS

Most persons who become infected with M tuberculosis mount a protective immune response and remain clinically well, the only evidence of infection being development of induration to the tuberculin skin test. Mter mycobacterial antigens

are introduced into an endosomic pathway in the macrophage, peptides of antigens

are subsequently presented on the macrophage cell surface on MHC II class

molecule. These complexes are then recognised by T cells bearing complementary

receptors that trigger the acquired T cell mediated immurie response and the

emergence of acquired resistance. In this regard, it seems likely that the cascade of

cytokines that are produced during the infection process are the key immunomodulators that drive such events and may mediate many of the clinical manifestations of tuberculosis.

When macrophages ingest M tuberculosis, they produce a characteristic

pattern of cytokines which may drive the immune response either towards

protection or suppression. IL-l is produced by M tuberculosis infected

macrophages and specific mycobacterial components such as LAM, and proteins

of 20 and 46KDa have been implicated in this (Wallis et al, 1990; Barnes et al,

1992; Zhang et al, 1993). IL-l is an endogenous pyrogen and may contribute to the fever that is characteristic of tuberculosis. Studies carried out by Platanias and colleagues (1990) established that IL-l induced macrophages to produce IL-6 and

TNFa, and stimulated T cell proliferation by upregulating T cell expression of IL-2 receptors and IL-2 production.

Murine as well as human mononuclear cells produce large quantities of

TNFa in response toM tuberculosis and also specific mycobacterial components

such as LAM and proteins of molecular weight 20, 44, 58 and 65 KDa (Moreno et

al, 1989; Wallis et al, 1993). Clinical and experimental data suggests that TNF

·contributes both to protection as well as immunopathology in tuberculosis. TNFa has been shown to enhance antimycobacterial activities of macrophages in vitro

(Flesch and Kaufmann, .1990). Granuloma formation in mice infected with BCG coincides with local accumulation of TNFa and neutralization of this cytokine has

been seen to interfere with the development of protective granuloma (Kindler et al,

1989). Takashima (1990) carried out studies on cytokine secretion by peripheral blood monocytes from patients with refractory and newly diagonised active tuberculosis. He found that patients with chronic refractory tuberculosis produced significantly lower amounts of TNFa than do monocytes from patients with newly diagonised tuberculosis. Other findings suggest that TNF plays a role in immunopathology. Excessive local production of TNF may cause marked tissue

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necrosis that is characteristic of progressive tuberculosis and its release in

circulation may contribute to systemic manifestations of the disease (Pisa et a/,

1990).

IL-6, a potent B cell growth and differentiation factor that induces antibody

production by activated B cells, is one of the prominent cytokines secreted by M tuberculosis infected macrophages. It may be responsible for the hyperglobinemia

that is characteristic of tuberculosis. IL-6 has been shown to reduce binding of

TNF to murine macrophages and to antagonize the antimycobacterial activity of

TNF in macrophages infected withM avium (Bermudez eta/, 1992). IL-6 has also ·

been shown to promote the intracellular and extracellular growth of mycobacteria

(Shiratsuchi eta/, 1991).

IL-8, is a cytokine with potent chemotactic potential for neutrophils, T

lymphocytes and basophils (Matsushima et a/, 1992). The secretion of IL-8 from.

M tuberculosis infected macrophages may contribute to the increased neutrophils

infiltration and T cell recruitment observed in tuberculous granulomas (Friedland

eta/, 1992).

IL-l 0 is an antiinflammatory cytokine that is produced by human and

murine macrophages exposed toM tuberculosis in vitro (Barnes eta/, 1992). IL-

10 may play a role in inhibiting the immune response toM tuberculosis and may

lead to the anergy and suppression observed in this disease. IL-l 0 has been

observed to inhibit cytokine synthesis and microbicidal activity of macrophages

(Fiorentino eta/, 1991; Oswald eta/, 1992).

Recent studies show that IL-12, produced by macrophages in response to

M tuberculosis infection may participate in the development of a protective

immune response. IL-12 has been shown to favor the development of precursor T

cells into Thl cells, which are thought to mediate resistance against mycobacteria

(Sypek et al,-1993; Yamamura et a/, 1991). Studies carried out by Boom and

coworkers (1992) have indicated that IL-12 enhances cytotoxicity by CD4+ T cells

against human macrophages pulsed with M tuberculosis. Zhang et a/ (1994)

suggested that IL-12 may serve as a growth factor during the early phase ofT cell

proliferation in response to mycobacterial antigens, by having a synergistic effect

along with suboptimal levels of IL-2 in enhancing lymphocyte proliferation.

It has been observed that, TGFf3 is constitutively overproduced by

monocytes from tuberculosis patients. Langerhan's giant cells and epitheloid cells

in tuberculosis granulomas also express mRNA for TGFf3 (Toosi eta/, 1995). TGF

f3 has been shown to inhibit cytokine synthesis by macrophages and down regulate

class II MHC expression (Espenik eta/, 1987; Czarniecki eta/, 1988). TGFf3 also

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inhibits IL-2 dependent T cell proliferation and IL-2 receptor expression. These

results indicate that TGFf3 inhibits antimycobacterial immune defenses and

facilitates mycobacterial survival.

Chemokines are low molecular weight polypeptides of 8-10 kDa and contain four Cysteine· residues at conserved positions. The superfamily has been

further subdivied into two distinct subfamilies, a and f3, based on whether the frrst two Cys residues are separated by one residue (C-X-C) or are adjacent (C-C). Chemokines are potent chemoattractants and regulate cellular composition of

inflammatory infiltrates by attracting specific subsets of leukocytes. The a and f3

chemokines are released by many cells after stimulation with proinflammatory

cytokines and LPS (Oppenheim et al, 1991). The f3 chemokines (MIP-lallf3,

MCP-112/3, RANTES) are chemotactic for monocytes or macrophages and some subsets of T cells, while a chemokines (NAP-2, MIP-2, IP-10) are mainly chemotactic for neutrophils. IP-1 0 is an unusual member of the a subfamily which

attracts monocytes and activated T cells rather than PMNs (Rhodes et al, 1995). Phagocytosis of Mycobacterium tuberculosis induces the secretion of IL-8 from THP-1 cells. IL-8 is a known chemotactic agent forT cells in vitro and hence may be important in granuloma formation (Friedland et al, 1992). Infection of murine

macrophages with M tuberculosis induces the rapid in vitro expression of genes encoding macrophage inflammatory protein-lex (MIP-la), macrophage

inflammatory protein-2 (MIP-2), interferon inducible protein (IP-10) and

macrophage chemotactic protein- I (MCP-1) (Rhodes et al, 1995).

CYTOKINES PRODUCED BY CD4+ T CELLS

The bulk of experimental data favors a dominant but not exclusive role for

CD4+ T cells in immune defense against tuberculosis. Predominance of Thl cells has striking effects on the manifestations of infection by intracellular pathogens.

Immunological resistance to mycobacterial infection in mice is mediated by Thl

cells. Lymphocytes from mice with immune resistance to M bovis produce high

concentrations ofiFNy and IL-2 (Huygen et al, 1992). Human T cells are capable

of exhibiting dichotomous patterns of cytokine production similar to those of

murine Thl and Th2 cells. The pattern of cytokine production in humans, correlates well with clinical manifestations of the disease. In patients with leprosy,

Thl cytokines such . as IFNy and IL-2 predominate in the skin lesions of tuberculoid leprosy patients who mount a resistant immune response toM leprae,

whereas Th2 cyokines IL-4 and IL-l 0 are prominent in lepromatous leprosy

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patients, with ineffective immunity and enormous bacillary burdens (Y amamura et

al, 1991). As an alternative approach to studing Th1 and Th2 profiles against M

tuberculosis antigens, CD4+ T cells and clones have been established from

PBMCs ofhealthy subjects vaccinated withM bovis BCG and those of pulmonary

tuberculosis patients. When tested with recall antigens like whole killed M

tuberculosis, 65 kDa heat shock protein and synthetic peptides, they demonstrated

a Th1 kind of response by producing cytokines like IL-2, IFNy and GMCSF. These

same mechanisms may also activate mediators of inflammatory response leading to tuberculous granuloma formation (Mustafa and Oftung, 1995).

HUMORAL IMMUNE RESPONSES

Although humoral immune responses do not play a central role in protective

immunity against tuberculosis, they have been under investigation primarily to

develop specific and sensitive serodiagnostics. Various groups have used different

antigenic fractions and probed them with sera from tuberculosis patients and

healthy controls in search of an antigen that may specifically recognise the patients. Studies carried out by Espitia et al (1989) found that while 50% of the

patients had antibodies to the 38 kDa protein in their sera, none of the control sera

recognised the protein in a Western blot. Bothamlay and coworkers (1992) used a

38 kDa based ELISA and reported that 85% of the sera from the patients with advanced tuberculosis and sputum smear positively to acid fast bacilli, had antibodies to the 38 kDa proteins while none of the control sera reacted to it. Only

about 15% of the smear negative patients had antibodies to the 38 kDa protein.

Antibodies to the nonprotein antigen LAM have also been found in

tuberculosis patients and not in healthy control subjects (Sada et al, 1990).

However, LAM is present in all mycobacteria and so it is inefficient in distinguishing between pathogenic mycobacteria and cross reactive environmental strains. Coates and associates (1989) studied the humoral responses of patients and healthy controls by using SDS PAGE and Western blot techniques. They reported

that a 10 kDa antigen was not recognised by any control sera, however only 10% of the patient sera had antibody against it. Although there have been report of other antigens- recognized differentially by patient and control sera the challenge

that still remains, is to devise strategies to dissect the human humoral response, in order to defme the species specific antigens or epitopes of M tuberculosis that are recognised only in individuals with active disease.

16

Review of Literature

DELAYED TYPE HYPERSENSITMTY REACTION

Observations made by Koch in 1891, paved the way for understanding the

cell mediated immunity (CMI) and delayed type hypersensitivity (DTH) observed

in tuberculosis. He observed that inoculation of tubercle bacilli produced different

results on the skin of a healthy guinea pig and that of a tuberculous one. The Koch

phenomenon was believed to be correlate in some way with resistance to tuberculosis, and that such a resistance was acquired and not innate. Subsequently, the Koch phenomenon was recognised to be a specific example of a generalized

mechanism that came to be called as delayed type hypersensitivity. It was in the year 1945, that Chase observed that tuberculin hypersensitivity in guinea pigs was

adoptively transferred to naive animals by cells as opposed to serum. Most individuals who become infected with M tuberculosis are able to contain the primary infection and develop a vigorous DTH response 2 to 4 weeks later. DTH

is associated with, but not identical to protective immunity. Although it has been

generally observed that patients with positive skin tests exhibit resistance to

exogenous reinfection, but many patients with severe tuberculosis have been

shown to have positive tuberculin test results. IFNy and IL-2 producing Thl cells

have been shown to enhance microbicidal activity of macrophages as well as augment DTH responses. At the present time, there is no conclusive evidence whether DTH producing T cells are the same as those producing protective immunity. Studies by Hussein et al (1987) have shown that some cloned T cell

lines mediate DTH but are not protective in vivo. Studies carried out by Orme

(1984) have also shown that separate cell populations are responsible for

transferring DTH and protective immunity.

FORMATION AND MAINTENANCE OF THE GRANULOMA

One of the first events that occurs after M tuberculosis is inhaled into the lungs is its interaction with the alveolar macrophages. The alveolar macrophages respond by phagocytosing the bacteria and by releasing a variety of

proinflammtory factors, including cytokines, bioactive lipids and oxidants. These factors may play a critical role in initiating the inflammatory response that may eventually lead to fibrosis and granuloma formation.

The initiation of granuloma formation involves multiple mechanisms and inflammatory mediators. Reactive oxygen metabolites appear to play an important role in the development of granulomas. Some of the most potent elements in hypersensitivity granuloma formation have been identified as early response cytokines such as IL-l and TNF. Cytokines and other ill defined factors released

17

Review of Literature

by the infected macrophage cause an increase in vascular permeability, which

leads to plasma leakage and clot formation under the action of procoagulant

factors (Chapman et al, 1986). Studies by Behling and colleagues have shown that

human macrophages can be induced to release procoagulant factors in response to

the mycobacterial product 'cord factor' (Trehalose 6, 6' dimycolate) (Behling et al,

1993). The net result is the formation of a fibrinous exudate in the alveoli, which

has been described as one of the early events of tuberculosis infection (Auerbach

and Dail, 1988). It has been speculated that the laying down of such a fibrin/

· fibronectin layer may provide a provisional matrix for immigration of

inflammatory cells.

Several lines of evidence suggest that specific cytokines are involved in

lung fibrosis. For example rodents treated with blocking antibodies to TNF show a

reduced fibrogenic response in hypersensitivity pneumonitis (Denis and Ghadirian,

1992). Studies carried out by Broeckelman and coworkers (1991) have shown that

increased amounts of TGFS are associated with human pulmonary fibrosis. It has

been observed that alveolar macrophages of tuberculosis patients produce high

amounts ofplatelet derived growth factor (PDGF) when exposed to supematent of

purified protein derivative (PPD) stimulated lymphocytes (Wangoo et al, 1993).

There is now increasing evidence to suggest that the cytokine network in

granulomatous inflammation causes fibroblasts to proliferate and secrete

extracellular matrix (Momex et al, 1994). Cytokines also induce the secretion of

hyaluronic acid (HA). Sampson and coworkers (1992) have presented evidence

that inflammatory cytokines such as TNFa, IFNy and IL-l stimulate the synthesis

ofhyaluronan by cultured lung fibroblasts. Galindo and associates (1975) showed

that when lung inflammation was induced by the injection of heat killed BCG, the

level of HA in the lungs were increased and which may play a part in macrophage

aggregation. Histological studies of the granuloma matrix have revealed the

presence of type III collagen (Peyrol et al, 1986), fibronectin (Kuroda et al, 1993)

and hyaluronic acid (Rochester et al, 1993).

The recruitment and activation of phagocytes into organised granulomas is

an essential component of resistance to a variety of microbial pathogens capable of

intracellular survival and is particularly important in mycobacterial infections. In

some cases, such a granuloma formation is strictly dependent on the induction of

antigen specific T cells and thus SCID mice infected with S. mansoni or L.

donovani fail to elicit the granulomatous response seen in their immunocompetent

counterparts (Kaye eta/, 1992). In contrast to this, studies carried out by Smith

and coworkers (1997) established the induction of granuloma formation in

18

Review of Literature

response to Mycobacterium avium m SCID mice, in the absence of antigen

specific immunity. Such a granulomatous response was· dependent on the

expression of TNFa and IFNy as depletion of either of these cytokines abolished

phagocyte recruitment, and resulted in the presence of a heavily infected cell

population in the absence of a surrounding inflammatory response; The reduced

inflammation observed following neutralisation of either IFNy or TNFa in SCID

mice was accompanied by a reduction in the in situ cytokine mRNA levels for IL-l

f3, IL-12, IL-10 and MIP-la demonstrating that in the case of M avium IFNy

and/ or TNF a are necessary for downstream expression of other cytokines

implicated in cell recruitment and likely to contribute to granuloma formation.

Increased mRNA expression and production of IL-l has been associated with the

development and severity of granulomas of tuberculosis and sacoidosis (Rolfe et

. a/, 1993 ). The regulation of IL-l during granulomatous lung inflammation may be

crucial for limiting the size of the lesion.

The essential role of IFNy in the control of mycobacterial infection has

been shown in mice with targeted mutation in the IFNy or IFNy receptor genes. In

such mice, infection with M tuberculosis led to caseous necrosis (Flynn et a/,

1993) or resulted in the formation of poorly differentiated granulomas on infection

with BCG (Dalton et al, 1993; Kindler et al, 1989). Studies by Garcia eta/ (1997)

have shown a synergistic role of TNF a and IFNy in the differentiation of

protective granulomas. They observed that granulomas of double deficient mice

were made up of large numbers of polymorphonuclear cells, eosinophils and cells

undergoing apoptosis, but without differentiated macrophages.

Studies carried out by Lopez-Ramirez eta/ (1994) have shown that in vitro

infection ofM tuberculosis H37Ra enhanced the expression ofiCAM-1 on THP-1

myelomonocytic cell line. Such an enhancement has also been observed in sarcoid

granulomas (Van Dinther-Janssen et a/, 1993) and in epidermal kertinocytes in

tuberculoid leprosy but not lepromatous leprosy (Sullivan et a/, 1991 ). Most et a/

( 1990) showed the importance of IFNy induced enhanced expression of LF A-1 in

the generation of multinucleated giant cells. It was observed that giant cell

formation could be completely blocked by antibodies to LFA-1 and ICAM-1. In

the alveolar macrophages away from the granuloma, none or weak expression of

ICAM-1 could be observed suggesting that such an expression was granuloma specific.

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Review of Literature

THE PATHOLOGY OF INFLAMMATION

Inflammation may ·be caused by soluble antigen, live organisms and

chemical or mechanical stress upon tissue. It is characterized pathologically by an

increased supply of blood to the affected area, increased capillary permeability

caused by retraction of the endothelial cells and infiltration of phagocytic,

monocytic and polymorphonuclear cells into the site of tissue insult. The initial

stages are known as the acute inflammatory reaction; when the process is

prolonged the inflammation may be sub-acute or chronic.

Acute Inflammation

The classical signs of acute inflammation are Rubor (redness), Calor (heat),

Tumor (swelling), Dolor (pain) and Functio laesa (loss of function). These are

microscopically explained by hyperaemia, exudation and emigration of

leukocytes. The hyperaemia in inflammation is associated with the well known

microvascular changes explained in Lewis's triple response: a flush, a flare and a

wheal. The flush and flare are physiological expressions of capillary and arteriolar

dilatation. A wheal results due to exudation or increased passage of protein rich

fluid through the vessel wall into the interstitial tissue. The increased protein

· passage occurs due to endothelial cell daniage by chemical mediators released

during injury. The loss of protein from the capillaries reduces the plasma osmotic

pressure, while the hyperaemia causes an increase in capillary pressure and the

resultant interstitial tissue protein leakage leads to an increased tissue osmotic

pressure. All these factors induce an enhanced filtration pressure resulting in a

local swelling or an oedema (Govan et al, 1981).

Chronic Inflammation

When the causative agent leading to an inflammatory episode, persists in

the tissue without clearance, a chronic inflammation results~ The further sequels to

this process are - appearance of lymphocytes and plasma cells, formation of giant

cells, reduction in the· number of polymorphs and the persistence of macrophages

at the site. It also results in hyperplasia, formation of new capillaries and fibrosis.

Chronic inflammation may also occur as a primary response with no preceeding

acute inflammatory reaction. A pertinent example of this is the formation of the

'tubercle' as the basic lesion in tuberculosis.

ADHESION MOLECULES AND INFLAMMATION

The salient features of a variety of inflammatory conditions, such as

infection, allergic disorders, autoimmune diseases or ischemia/reperfusion injury is

the association of infiltrating leukocytes. These extravasating leukocytes often

20

Review of Literature

contribute to the pathogenesis of the underlying disease. However, it needs to be appreciated that leukocyte recruitment is also critical for host defense, leading to

clearance of the inciting factor or infection. The maintenence of leukocyte

recruitment during inflammation requires intercellular communication between

infiltrating cells and the endothelium. The functions of adhesion molecules in

inflammation and the regulation of their expression are now known to be of paramount importance in the outcome of any inflammatory response. This

movement requires the action of specific signaling molecules, that affect the

expression of, or function of members of three families of adhesion molecules

present on the endothelium or leukocyte, namely, the Selectins, the lntegrins and the Immunoglobulin (Ig) superfamily (Table 1 ).

Selectins Selectins are a family of carbohydrate binding proteins or lectins which

play a pivotal role in the early stages of lymphocyte recirculation and

inflammatory responses (McEver eta/, 1995). The three known selectins, termed L, E and P-selectins have domains homologous to other Ca2+ dependent lectins.

Each molecule is known to contain a lectin like amino terminal domain, an epidermal growth factor repeat and a discrete number of modules ( ~60 amino acids

each), similar to those found in certain complement binding proteins (Bevilacqua et a/, 1993). L-selectin is constitutively expressed by most circulating

lymphocytes, neutrophils and monocytes, and appears to be involved in the

binding and extravasation of leukocytes to inflamed sites (Spertini eta/, 1991). It

binds to 50KDa and 90KDa glycoproteins from lymphoid tissue which have been

shown to contain sulphated, sialylated and fucosylated 0-linked oligosacchrides. Treatment of the ligands with sialidase prevents recognition by L-selectin. The 0-

linked glycans in the 50KDa ligand, termed as Glycosylation dependent Cell

Adhesion Molecule-1 (GlyCAM-1), are heterogenous with respect to both charge and size. The 90KDa molecule is identical to CD34, and the broad distribution of this molecule on endothelial cells suggests a role as an inducible ligand for L­selectin (Lasky et a/,1992; Baumhueter eta/, 1993).

P-selectin is stored in the a granules of platelets and. Weibel Palade bodies

of vascular endothelial cells, and their cell surface expression is induced within minutes of stimulation with thrombin or oxygen radicals (Patel eta/, 1991). It has been shown to bind to a single trace sialoglycoprotein in the extracts of human neutrophils. The predicted amino acid sequence for this molecule, termed, P­Selectin Glycoprotein Ligand-1 (PSGL-1) contains three potential sites for N­linked o1igosaccharides and multiple sites for 0-linked glycosylation. Sialidase

21

TH- '::f-763

Adhesion Molecule Subunit CD Designation Ligand

12 Suoerfamilv CD2 CD2 LFA-3 LFA-3 CD 58 CD2 ICAM-1 CD 54 LFA-1, Mac-1 ICAM-2 CD102 LFA-1, Mac-1 ICAM-3 LFA-1 VCAM-1 CD106 VLA-4 NCAM CD 56 NCAM, Heparan Sulfate PECAM-1 CD31 CD31, a." 13,

Inte2rin Family VLA-1 a. 13 CD49a/CD29 Laminin, Collagen VLA-2 a., 13. CD49b/CD29 Laminin, Collagen VLA-3 a.3 131 CD49c/CD29 Laminin, Collagen,

Fibronectin VLA-4 a. 131 CD49d/CD29 VCAM-1, Fibronectin VLA-5 a.s 131 CD49e/CD29 Fibronectin VLA-6 a." 13 CD49f/CD29 Laminin

a." 13. CD51/CD29 Fibronectin, Vitronectin LFA-1 a._l 13,., CD11a/CD18 ICAM-1 ,ICAM-2,ICAM-3 Mac-1 a.M 132 CD11b/CD18 ICAM-1, ICAM-2, LPS,

Fibrinogen, C3bi p150,95 Uy tl, CD11c/CD18 Fibrinogen, C3bi, LPS

a." B,., /CD18 ICAM-3

a.v 133 CD51/CD61 Vitronectin, Fibrinogen, Fibronectin, Factor VIII, Thrombospondin

Gpllb/llla a.llh 13'1 CD41/CD61 Fibrinogen, Factor VIII

a." 13. CD49f/ Laminin, Kalinin

a.~ J3s CD51/ Vitronectin

Uv 13" CD51/ Fibronectin

a.4 137 Cd49d/ MAdCAM-1, VCAM-1, Fibronectin

Up 13.., CD103/ E- cadherin

Selectin Family L- selectin CD62L GlyCAM-1, MAdCAM-1,

CD34 E- selectin CD62E ESL-1, PSGL-1, CLA P- selectin CD62P PSGL-1 '

Table 1. The Adhesion Molecule Families

Review of Literature

treatment of the native ligand from myeloid cells has been shown to abolish its

interaction with P-selectin (Moore et al, 1994).

E-selectin is fundamentally involved in inflammatory responses and

facilitates the recruitment of neutrophils and monocytes, which constitutively

express ligands for the selectin. It is expressed transiently on endothelial cells

within 2-6 hrs following activation by proinflammatory cytokines (Bevilacqua et

al, 1987). A recent addition to the selectin ligand repertoire is the Mucosal

Addressin Cell Adhesion Molecule (MAdCAM-1) which contains two N-terminal

domains with homology to the immunoglobulin superfamily members, followed by

a mucin like domain and ending with an IgA like domain. This molecule has been

shown to support the binding ofL-selectin, as well as a4137 integrin binding (Berg

et al, 1993; Berlin et al, 1993).

Integrins

lntegrins are al3 heterodimers that play important role during development

and in adult organisms. The ability to interfere with integrin functions using

antibodies or peptides offers opportunities for therapeutic interventions in diseases

such as thrombosis, inflammation and cancer.

The a subunit of integrins vary in size between 120-180KDa and are non­

covalently associated with a 13 subunit (90-110KDa). Most integrins are expressed

on a wide variety of cells and most cells express several integrins. There are 8

known 13 subunits and 16 known a subunits. Although in theory, there could occur

more than 100 heterodimers, the actual diversity is much restricted (Hynes, 1992;

Stewart eta/, 1995). The integrins can be subdivided into classes based on the 13

subunit, which can be associated with one or more a subunits. The most widely

distributed integrins belong to the 131 class. They are also known as the Very Late

Antigens (VLA), and most of them interact with the extracellular matrix (ECM)

proteins. The second class of integrins are leukocyte specific receptors and consist

of one of the three a subunits ( aL, aM or aX) complexed with the 132 subunit.

The cytoadhesins, alllbl33 and avl33 constitute the third class of integrins. A wide

variety of proteins serve as ligands for integrins, among them are ECM proteins,

plasma proteins and cell surface receptors.

Cell adhesion may be strengthened by the ability of individual integrins to

display overlapping ligand recognition. There are numerous examples of

regulation of integrin function. The regulation of integrin expression may be

evident at the level of developmental differentiation (Miller et al, 1986), activation

dependent enhancement of expression (Shimizu et al, 1990) or cytokine dependent

modulation of expression (Ignotz et al, 1987). Ligand binding, and for some

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Review of Literature

' integrins, subunit association is divalent cation dependent. For instance, a4~ 1

promoted eosinophil adhesion to VCAM-1 requires the presence of both Ca2+ and Mg2+ (Dobrina eta/, 1991). Studies carried out by O'Toole eta/ (1990) suggested

that activation may be accompanied by a conformational change in the integrin

which can be detected immunologically. The expression of integrin neoepitopes

upon activation by Mn2+ has been found to be important for LFA-1 dependent

adhesion (Lub eta/, 1995). The ligand specificites of a particular integrin may be

controlled by cell type specific modulation. The most striking example of this is

seen in the case of a2 ~ 1 which is a collagen receptor on platelets and both a collagen and laminin receptor on endothelial cells. (Kirchhofer eta/, 1990). The

most versatile example of modulation of integrin function is seen in the case of

affinity modulation of the ~2 integrins in response to a variety of stimuli, primary among them being the chemokines (Arnaout, 1990; Dustin and Springer, 1989).

The Ig Superfamily

The Ig superfamily accounts for 50% of all cell surface molecules displayed on leukocytes. This evolutionary success story is probably due to the stability of

the Ig domain which is able to resist the harsh proteolytic and oxidative

environment of the extracellular milieu. Ig receptors have evolved to serve several

functions such as receptors for growth factors, receptors for the Fe region oflg and

as adhesion molecules which now seems to be the function of the majority of these

molecules (Holness and Simmons, 1994).

The packing and orientation of domains in the Ig superfamily molecules seems to be remarkably uniform and yet there exists a striking diversity of modes

of interaction amongst individual members. Such interactions although diverse in

an individual sense, seem to follow two basic patterns of recognition: heterotypic

binding mediated by the N terminal domain and homotypic binding mediated by

'internal' domains and requiring antiparallel allignment of interdigitated molecules.

The heterotypic mode is important in the interaction of integrins with the Ig

superfamily members. Important examples of such interaction can be observed in the binding ofiCAM-1 with LFA-1 'and/or Mac-1 (Diamond eta/, 1991); VCAM-1 with VLA-4 and a4~7 (Ruegg eta/, 1992); and MAdCAM-1 with a4~7 (Berlin et a/, 1993). The homotypic mode of interaction may range from a simple head to head interaction as seen in the case of CD66-CD66 binding ; to the more complex interdigitating interaction required for CD31 or NCAM. (Rao eta/, 1992).

The Adhesion Cascade Dogma: Pathways and preferences The recruitment of effector cells to the site of antigenic insult or injury, is

one of the most dramatic responses to tissue damage, and of any inflammatory

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Review of Literature

response. It is also central to the normal physiological trafficking of leukocytes. Both processes occur due to selective leukocyte-endothelial recognition. The

'selectivity' of recruitment is probably the key distinguishing feature of one kind of

inflammation versus another (Springer, 1995). Patients deficient in LFA-1 due to

mutations in the ~2 integrin gene, were the earliest evidence that adhesion molecules were required for leukocyte extravasation in vivo (Anderson and

Springer, 1987; Larson and Springer, 1990). Neutrophils in these patients fail to

cross endothelium and hence do not accumulate at sites of inflammation and, in

vitro are unable to adhere and transmigrate through activated endothelial cells.

The Adhesion Cascade Leukocytes transmigrate. into tissue under three sets of guidelines. First,

naive T and B cells have a selective 'homing' pattern of migration via ~e high endothelial venules (HEY) into the secondary lymphoid tissue, where they might encounter processed antigen on antigen presenting cells (APC) and subsequently

get activated (Picker, 1992). Second, lymphoblasts and other antigen activated

leukocytes display tissue restricted migration to extra lymphoid organs and sites

such as mucosal epithelium or skin (Foster et a/, 1990; Saltini et a/, 1990). By

varying the expression of homing receptors and counter receptors, and by allowing

their sequential cooperation in variations of the multistep theme, the immune system can construct many specific homing pathways using relatively few distinct molecular components. Recent data from various laboratories supports the contention of organ specific migration of 'naive' and 'memory' T cells (Westermann

and Pabst, 1996; Butcher and Picker, 1996). Finally, lymphocytes as well as

neutrophils and monocytes migrate into inflamed tissues in response to localized

stimuli. Granulocytes and monocytes normally do not recirculate back into blood

(Chisholm et a/, 1993). The nature of the inflammatory stimulus determines

whether lymphocytes, monocytes, neutrophils or eosinophils predominate; and thus exercises specificity in the molecular signals that control the traffic of particular leukocyte classes. With the establishment of the role of adhesion

molecules in leukocyte infiltration, several workers began to dissect out the steps

that occured during the adhesion cascade. It soon became clear that leukocyte migration required three major steps; which consist of rolling, tight adhesion and extravasation (Fig. B). Three families of adhesion molecules were implicated in these events. Studies on the basic molecular mechanism of the inflammatory response support a model in which a cascade of events brought about by the sequential action of different adhesion molecules, lead to the transmigration of leukocytes across the endothelium (Springer, 1995). In the first step, leukocytes

24

----- Selectins & lntegrins----­

lntegrins--------

Endothelium

Lymphocyte

@

~ Activation

~ethering Ar~est & Rolli

-------Chemoattractants ---­lntegrins-

Blood Stream

Extravasation

Fig. B. The Adhesion Cascade

Review of Literature

within the venular lumen interact with endothelial cells via the action of activation

independent leukocyte receptors with regulated endothelial ligands or counter

receptors. This interaction, mediated by selectins or a4 integrins, leads to transient tethering and subsequent rolling of the leukocyte on the endothelium, thus

exposing it to activators released by the endothelium or underlying tissue (Berlin

eta/, 1995). Antagonists ofL and E-selectin have been shown to inhibit neutrophil

and monocyte influx in response to inflammatory agents (Picker and Butcher,

1992; Mulligen et a/, 1991 ). At sites of inflammation, leukocytes first attach to the vessel wall in a rolling interaction and then become arrested and firmly adherent at

a single location on the vessel wall before diapedesis (Cohnheim, 1889). This process was fully reconstituted with purified constituents of the endothelial surface by Lawrence and Springer (1991). They observed that at physiological shear

stress, neutrophils attached and formed labile rolling adhesions on phospholipid

bilayers containing purified P-selectin but not ICAM-1. At physiological shear

stress, if both P-selectin and ICAM-1 were present; resting neutrophils attached

and rolled identically as on P selectin alone. However, when chemoattractant was added the rolling cells arrested, spread and fmnly adhered through intergrin/ICAM-1 interaction. Chemoattractants do not enhance interactions with P

selectin monolayers alone. This suggested that selectins were required prior to

chemoattractant stimulated integrin adhesion. This dogma of Selectin~Integrin/Ig

superfamily sequence was shattered when it became clear that at least some

integrins participated in the rolling event. Studies have shown that the integrin VLA-4 supports tethering and rolling on VCAM-1 (Alan et a/, 1995) and the

integrin a4f37 participates in rolling during lymphocyte homing to Peyer's patches (Bargatze eta/, 1995). Recent studies by DeGrendele and coworkers (1996) have

provided data to suggest that CD44 and its principal ligand hyaluronate mediate

rolling of T cells on endothelium under physiologic flow and that this might

represent a novel lymphocyte endothelial cell primary adhesion pathway.

In the second stage, cellular arrest occurs under conditions of flow when a4 integrins bind their Ig superfamily ligands after becoming activated under the

influence of inflammatory mediators and chemoattractants (Lucinskas eta/, 1994). Recent studies have elegantly elucidated the molecular regulation of adhesion molecule interactions under high flow conditions. They have revealed that the tethering and rolling steps are highly dependent on shear forces (Finger et a/,

1996). L-selectin has been shown to be concentrated on the tips of lymphocyte microvilli (the site of initial contact under flow), a feature that it shares with other tethering recptors ( a4 integrins and probably PSGL-1 ), and which dramatically

25

Review of Literature

enhances the efficiency of receptor engagement under shear force (von Adrian et

a/, 1995). In the next stage, leukocytes use the P2 integrins LF A-1 and Mac-1 to

fmnly adhere to the endothelium, flatten and transmigrate into the tissue via

ligation with ICAM-112. In some circumstances, a4P 1 can accomplish this goal

independently of the P2 integrin (Issekutz and Issekutz, 1995). The CD31 (PECAM -1) molecule has also been implicated in the 'diapedesis of leukocytes

(Muller eta/, 1993a).

The Recruitment Of Cells . Studies implicate the involvement of at least three major adhesion pathways

in the migration ofT lymphocytes. These pathways are mediated by the integrin-Ig superfamily ligand pairs such as LFA-1/ICAM-1, VLA-4NCAM-1 and VCAM-11

a4J37. Increasing evidence also supports a role for MAdCAM-lla4J37 interaction

in cell adhesion (Bradley and Watson, 1996). LFA-1 mediated adhesion and costimulatory functions occur through interactions with the counter receptors ICAM-1 and ICAM-2. Induction of ICAM-1 on endothelium and other cells by

inflammatory cytokines may increase cell-cell interactions and leukocyte

extravasation at inflammatory sites, whereas, constitutive expression of ICAM-2 may be important for leukocyte trafficking in uninflamed tissues, as in lymphocyte

recirculation. Mac-1, which is expressed primarily on cells of myelo-monocytic

lineage, recognizes ICAM-1 and plays a primary role in the recruitment of these cells (Xie et a/, 1995). The adhesion cascade, by virtue of its functional attributes,

permits a great deal of selectivity in terms of leukocyte subsets and adhesion molecule usage. This selectivity is largely dependent on the nature of the cytokines

and chemokines released during a given inflammatory episode. Cytokines

differentially modulate the expression of a number of adhesion molecules on endothelial cells. The expression is regulated by the inducing cytokine(s), the time

of exposure and the type of environmental cues from neighbouring cells and extracellular matrix. The upregulation of selectins and their ligands, is largely dependent on cytokine, histamine and thrombinrelease (Hogg and Berlin, 1995).

IL-lP and TNFa have been shown to upregulate ICAM-1, VCAM-1 and E­

selectin expression on endothelial cells (Pober et al, 1986). IFNy and IL-4 selectively upregulate ICAM-1 and VCAM-1, respectively (Issekutz, 1990). Further, different cytokines induce the release of different chemokines. For eg: IFNy, but not IL-4, can induce the secretion of RANTES by endothelial cells (Marfaing-Koka et a/, 1995). Chemokines in turn can exert specificity in the

subset of leukocyte recruited, for eg: MIP-1 p recruits naive CDS+ T cells by

26

Review of Literature

activating VLA-4 while RANTES selectively recruits memory T cell subsets

(Tanaka et a/, 1993). Integrin activation is an important step in leukocyte extravasation and is

fmely regulated by a number of mechanisms. In vitro activation of T lymphocytes

through CD3/TCR complex enhances T cell adhesion to endothelium and is

mediated by an LFA-1/ICAM-1 pathway (Dustin and Springer, 1989). Stimulation

of lymphocytes with phorbol esters, calcium ionophores and mitogens rapidly and transiently increases integrin avidity. In vivo antigen activation during an immune

response has been found to result in enhanced adherence of lymph node T cells to

endothelium (Issekutz, 1991 ).

MODULATION OF ADHESION MOLECULES EXPRESSION IN DISEASES

The cell surface expression of many adhesion molecules is upregulated

following activation during a microbial invasion or an inflammatory response. It

may be responsible for both cell activation and migration during a protective

immune response on one hand, and exacerbating the pathology of the disease in other cases. In most animal models of inflammation, E selectin is expressed early

and correlates with neutrophil influx, whereas ICAM-1 and VCAM-1 expression

increases with a more prolonged time course and correlates with mononuclear leukocyte infiltration. Increased levels of LF A-1 and ICAM-1 have been observed

in alveolar macrophage of patients of pulmonary sarcoidosis, which is a chronic disease characterized by mononuclear cell alveolitis and granuloma formation

(Melis eta/, 1991). In a study carried out by Grober and colleagues (1993) the cell surface expression of Mac-1 was found to be upregulated on CD14+ blood

monocytes from patients of rheumatoid arthritis, and correlated significantly with

synovial infiltration of mononuclear cells. ICAM-1 has been found to play a

crucial role in granuloma formation in schistosome infected mice (Lukacs et al,

1994), and studies by Langley and Boros (1995) have shown that T lymphocyte

responsiveness in murine schistosomiasis is dependent on the adhesion molecules ICAM-1, LFA-1 and VLA-4. The accumulation of leukocytic infiltrates in perivascular tissues is a key step in the pathogenesis of Lyme disease, a chronic inflammatory disorder caused by Borrelia burgdorferi. It has been reported that B.

burgdorferi causes an upregulation of E-selectin, VCAM-1 and ICAM-1 on endothelium and also enhances neutrophil migration across activated endothelium (Sellati eta/, 1995). In a recent study, Rocha et al (1997) have reported that the

intense inflammatory colitis caused by C. difficile toxin A is due to its ability to induce the release of neutrophil chemotactic factors from macrophages.

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Review of Literature

During the emergence of the acquired response to mycobacterial infection, a number of cytokines and chemokines are produced. Infection with M tuberculosis induces a prolonged increase in the surface expression of ICAM-1 on

THP-1 cell line (Lopez-Ramirez et al, 1994). This sustained increase in ICAM-1

expression may facilitate the recruitment of cells needed to maintain the structure

of granuloma, or assist the activation of T cells. It has been observed that

microorganisms which can be completely degraded by macrophages in culture,

evoke ·only transient acute inflammatory responses in vivo and only those that are

resistant to degradation.induce granuloma formation (Adams, 1976). The induction

of adhesion molecule expression on immune cells might have important consequences in the development of the inflammatory response, as skin biopsies from patients of lepromatous leprosy (characterized by T cell anergy and low CMI) have been shown to lack ICAM-1 expression on keratinocytes (Moncada et

al, 1993).

· Acute inflammatory reactions are characterized by the local accumulation

of leukocytes at the site in response to stimulus generated by resident tissue cells.

A number of cytokines and chemotactic factors have been implicated in causing cell migration in in vitro cell adherence and migration assays. The in vivo role of a

few of these factors has been established using animal models. Intradermal injection of TNFa to baboons. caused upregulation of VCAM-l on vascular endothelium and resulted in the recruitment of T cells to the site of injection

(Briscoe et al, 1992). Another group observed that intradermal administration of

NAP-1 (Neutrophil activation protein) to human subjects caused a predominantly

neutrophilic infiltration (Leonard et al, 1991 ). Such a model has also been used to

study differences in cell recruitment to the parenchyma of CNS and to the skin

(Andersson et al, 1992).

MOLECULAR BASIS OF ENDOTHELIAL CELL ACTIVATION

Vascular endothelial cells function as a dynamic interface between the elements of blood and the underlying tissues. In the basal state, endothelial cells

oppose thrombus formation and leukocyte and platelet adhesion. The elaboration of proinflammatory cytokines and the generation of coagulation cascade products

induce .. both early and delayed changes in endothelial cell morphology and function at sites of vascular injury and inflammation. Rapidly occuring events, termed 'type I activation', include reversible cell shape change and retraction, secretion of von Willebrand factor and tissue plasminogen activator, surface expression of P selectin and formation/release of prostacyclin, platelet activating

28

Review of Literature

factor and nitrous oxide. These early events proceed rapidly and independently of protein synthesis and are typically associated with transient elevations of Ca2+. In

contrast, delayed and often sustained phenotypic changes of endothelial cells,

termed 'type II activation' are regulated at the level of transcription and occur in

response to proinflammatory cytokines, bacterial LPS, or contact with immune

effector cells. These changes consisit of increased de novo expression of a diverse

array of proteins including adhesion molecules that promote leukocyte

sequestration and diapedesis, proinflammatory cytokines, granulocyte CSF and

proteins of the hemoststic and fibrinolytic pathways (Anrather et al, 1997).

The genes encoding many of these proteins contain, within their 5'

regulatory regions, motifs that can be recognized by transcription factors; prominent among them being the ones for NFKB, AP-1, Sp1 and the interferon

response element (IRF -1 ) .. The best characterized promoter in endothelial cells is

that for the E selectin gene. Canonical p50/65 NFKB heterodimers are able to bind

to three distinct sites in the positive regulatory domain. An additional element is

the CRE/ ATF like binding site that is mainly occupied by ATF2 dimers (Activating Transcription Factor, a member of the basic leucine zipper family),

which plays a major role in the cytokine inducibility of the gene (Collins et al,

1995).

The VCAM-1 promoter shows a tandem ofNFKB binding elements that are

necessary for cYtokine mediated expression, and are targets for the binding of classical p50/65 heterodimers or p65 homodimers. More recently, other elements

have been described, including an IRF -1 and an Sp 1 binding site. Finally the

HMG 1(Y) protein [High Mobility Group Protein 1(Y)], appears to facilitate NFKB

and IRF-1 binding, and seems to be a crucial element for the cytokine inducibility

of the gene. The ICAM:-1 promoter shows three functionally relevant elements: an

atypical NFKB site that mediates binding of p50/65 heterodimers 'as well as c­

Rel/p65 and p65 homodimers; an element that mediates binding to p91 (STAT1) in response to IFNy and a C/EBP element that mediates binding to a combination of

factors (C/EBPa and~) (Neish et al, 1995).In vivo, the ability of TNF to activate other transcription factors, such as AP-1 and ATF may be important in the

generation of the overlapping but distinct patterns of gene expression by the endothelial cells (Collins et al, 1995).

Unlike most other transcriptional activators, members of the NFKB/Rel family of proteins reside in the cytoplasm and consequently must be translocated to the nucleus in order to exert their effects. In the quiescent state, NFKB exists as an inactive complex composed of 50(p50) and 65kDa (p65/Rel A) subunits

29

Review of Literature

noncovalently associated with IKB proteins which mask the NFKB nuclear

localization signal and thus prevent the translocation step. Phosphorylation and

subsequent ubiquitin/proteasome dependent degradation of the IKB molecules are

thought to be rate limiting steps in NFKB dependent gene activation. Inhibition of

NFKB activation by antioxidants such as pyrrolidinedithiocarbamate; or inhibition

of IKB proteolysis by selective protease inhibitors prevents the activation of NFKB

and the transcriptional upregulation of a variety of genes associated with type II

endothelial activation (Baeuerle and Baltimore, 1996). The redox status of the cell

plays an essential role in regulating signal transduction, transcription factor

activity and expression of cell surface molecules since reactive oxygen

intermadiates have been reported to act as common messengers in the activation of

several transcription factors such as NFKB and AP-1. This has been demonstrated

by the· inhibitory effect of antioxidant agents (possibly through scavenging and

inactivating reactive oxygen intermediates) on the induction of NFKB and by the

functional repercussions derived from these effects (Schreck et al, 1992; Muller et

al, 1993; Mantovani et al, 1997) (Fig. C).

The central paradigm ofNFKB activation, which involves the removal of IK

B proteins from a cytopHtsmic complex with NFKB, has been subjected to a

. detailed molecular analysis. The major pathway used by a variety of stimuli to

activate NFKB involves the phosphorylation of IKBa at its regulatory N terminus

on Ser 32 and 36, an event which leads to subsequent proteasome mediated

degradation of the inhibitor. It has been shown that mitogen activated kinase

pp90rsk can phosphorylate IKBa on Ser 32 in response to PMA, okadaic acid and

LPS but not TNF or HTLV-Tax suggesting that this kinase may be predominantly

used by mitogenic signalling pathways. Phosphorylation of IKBa on Tyr 42 in

response to pervanadate treatment ofT cells or reoxygenation of hypoxic cells has

been shown to cause NFKB activation without causing proteolytic degradation of I

KBa (Baeuerle and Baltimore, 1996).

Elevation of intracellular cAMP levels inhibit the induction of a distinct set

of NFKB regulated genes. In endothelial cells, elevation of cAMP inhibits

cytokine induction of E selectin and VCAM-1 expression (Pober et al, 1993).

Studies carried out by Ollivier et al (1996) have shown that elevation of

intracellular cAMP and· activation of Protein Kinase A in endothelial cells and

monocytic cells inhibits NFKB mediated transcription. Different protein kinases

may play a role in NFKB activation depending on the cell type studied. A number

of studies have analyzed the role of Protein Kinase C (PKC) and Protein Tyrosine

Kinase (PTK) in the modulation of adhesion molecule expression on endothelial

30

Nucleus Cytoplasm

A20 gene

•• I ~ .· \ ~ e \.e

Glucocorticoids

(e • • 1-· -----,~

~-a gene

<±) NO

(NF-KB) >-~ Activating signals

~ j. ~:B <±>

Kinases e

~ ..

· ...

~ As~rin/

0 0 ~ Serine protease --- ~F-KB) inhibitors

CD@ e Degradation @ ,__ _____ _

Antioxidants

production

(f) • "\

6\

Fig. C. Regulation of NFKB in Endothelial cells

Review of Literature

cells by employing specific activators or inhibitors. It appears that the mediation of

a specific pathway is dependent on a number of factors such as the adhesion

molecule being studied, the activating factor and the duration of treatment. Sung et

al (1994) observed that cytokine (TNF, LPS) induced ICAM-1 expression on HUVEC was inhibited by the PKC inhibitor, H7. Another group (Eissner et al,

1994) reported that TNF and LPS induced ICAM-1 expression on a human

endothelium derived cell line (EaHy 926) was unaffected by inhibition of PKC. In contrast, ICAM-1 expression induced by IFNy and IL-l was significantly reduced

by blocking this pathway. May et a/ (1996) investigated the effects of PTK

inhibitors on cytokine induced adhesion molecule expression on HUVEC. They

observed that genistein and Herbimycin A could inhibit TNFa induced expression

ofE selectin, ICAM-1 and VCAM-1, while bisindolylmaleimide (a PKC inhibitor)

had no effect.

METHODS USED IN THE QUANTITATION AND ANALYSIS OF LEUKOCYTE

ENDOTHELIAL INTERACTIONS

Amongst the earliest methods used to study leukocyte-endothelial

interactions was the cannulation of the thoracic duct (Spry, 1972). During the

same period in vitro assays, that were rapid and permitted a more precise

understanding of the molecular mechanisms were developed. The most practical

and effective of these being the Stamper and Woodruff assay (1976). The principle

that operated behind this and later assays to come, was that tissue sections of lymph nodes or cultured endothelial cells provided an ideal substrate to gauge the

interactions of endothelial cells with lymphocytes under conditions of activation.

Subsequently, several elegant assays using endothelial cells cultured on a bed of

extracellular matrix proteins were used not only to study lymphocyte adhesion, but

migration as well. A drawback of the Stamper Woodruff assay was the inability to

study lymphocyte migration. Lymphocyte migration may be studied using

transwell tissue culture inserts which enable the growth of endothelial cells on the

polycarbonate filter of the transwell. Both radioactive chromium and fluorescent

dyes are used to label lymphocytes to permit an accurate and rapid quantification

of lymphocyte adhesion and extravasation. While several workers use these assays even today, some feel that these assays are not adequate and do not accurately represent the in vivo scenario.

The answer to these problems was found in the usage of flow chambers

which consist of two parallely arranged stainless steel or glass plates, separated by a silicanized gasket. The bottom of the plates can be coated with soluble adhesion

31

Review of Literature

molecules or can be plated with endothelial cells. Lymphocytes suspended in warm medium are then perfused through the flow chamber at defmed flow rates using a syringe pump. The temperature of the whole apparatus is maintained at 3 7° .

C. The chamber is mounted on an inverted phase contrast microscope and the

entire perfusion period is recorded on a videotape by a video camera and a VCR.

Using automated PC based image analysis programs both the number of adherent

lymphocytes and rolling velocities can be determined accurately.

Several phenomena that regulate inflammatory events have been studied in this way including the specific adhesion pathways used by different subsets of T cells (Luscinskas et a/, 1994). Recently, Finger et a/ (1996), have used flow chambers to show that a threshold hydrodynamic shear force is required for the optimal binding of L selectin to its ligand. This brings out an inherent problem

associated with static assays which do not permit optimal conditions for all the

steps of the adhesion cascade. However, static assays are useful in assaying the

interactions of integrins with their ligands, particularly when the analysis of arrest and tight adhesion is to be studied. While flow assays are ideal to study selectin or integrin interactions with their receptors during tethering and rolling, the binding

of receptor ligand during rolling does not trigger activation of integrin, since lymphocytes attach and roll during flow on purified ligands for selectins

identically, whether or not Ig superfamily molecules are present on the substrate, and an additional stimulus is required before they attach firmly (Springer, 1995).

This second· step of arrest and tight adhesion can be studied using s!atic assays, as

shear force does not seem to affect this step. This is proved by the fact that several molecular mechanisms have been accurately defmed by the static assays. This is particularly true of integrin/ Ig superfamily interactions (Anderson and Springer,

1987; Larson and Springer, 1990; Shimizu et a/, 1990). Recently, a novel

leukointegrin ad~2 was discovered and· shown to preferentially bind ICAM-3 using static assays (Vander Vieren eta/, 1995). Static assays have also been used to address issues of chemokine activation of integrins and chemokine induced migration (Tanaka eta/, 1993; Schall eta/, 1993).

32

Materials and Methods

MATERIALS AND METHODS

3.1 MICE BALB/c By J inbred mice were originally obtained from Jackson Laboratory, Bar

Harbor, Maine, USA. They were bred and maintained under pathogen free

conditions in the Small Animal Facility of the Institute.

3.2 MYCOBACTERIA Mycobacterium tuberculosis strains H37Ra and H37Rv were originally obtained

from Trudeau Institute, Saranac Lake, New York, USA and maintained on

Lowenstein-Jensen slants in the laboratory.

3.2.1 BACTERIAL MEDIA: Nutrient agar, Middlebrook 7H9 broth, Mycobacteria

7Hll agar, ADC and OADC enrichments, Lowenstein-Jensen medium and Bacto­

glycerol were bought from Difco Laboratories, Detroit, USA.

3.2.1a Nutrient Agar: 23g of dry powder was dissolved in I liter of glass distilled

water and sterilized by autoclaving for 20min. at a pressure of 15 psi. The medium

was then poured into sterile petridishes and allowed to set.

3.2.1b Middlebrook 7H9 Medium: 4.7g of the commercially available powder

was dissolved in 900ml of glass distilled water, containing lml Bacto-glycerol and

0.05% Polysorbate 80. After sterilizing at 15 psi for 20min., the medium was

allowed to cool to a temperature of 45-50°C and lOOml of ADC enrichment was

added to it (final pH 6.6±0.2).

3.2.1c Mycobacteria 7H11 Agar: 21g of the commercia1ly available powder was

dissolved in 900ml of glass distilled water containing 5ml glycerol and the

medium was sterilized. 1 OOml of OADC enrichment was added to it when the

medium had cooled to a temperature of 45-50°C.

3.2.2 MAINTENANCE OF MYCOBACTERIAL CULTURES: The stock culture of

mycobacteria was maintained on L-J slants. The culture was streaked on 7Hll

plates and colonies were inoculated in 7H9 broth for bulk culture. The culture was

grown at 37°C under shaking condition and was harvested after 14 days. The

mycobacteria were washed twice in sterile PBS and dispersed with mild sonication (three pulses of 1min. each at 50% output, using the regular probe of Branson B-30 sonifier, USA) to minimize clumping of bacteria. Purity of the culture was assessed by streaking on nutrient agar plates to confirm the absence of other

33

Materials and Methods

bacilli. Mycobacteria were stained by Ziehl-Neelson method (Kubica, 1984) and

were counted by using the ring slide method of Hart and Rees (1960). The viable units were determined by counting the colony forming units (CFU) at appropriate

dilutions of the culture inoculated on 7H 11 plates.

3.2.3 ZIEHL-NEELSON METHOD FOR STAINING ACID FAST BACILLI: Appropriate

dilutions of the mycobacterial suspension were smeared evenly on a glass slide

and air dried. After gentle heat fixation, the smear was flooded with 1% carbol

fuchsin and flamed till smoke was seen to rise. The excess stain was washed off

with tap water and the smear was decolorized with acid alcohol (0.5% HCl in 70%

alcohol). The slide was counterstamed with methylene blue for 1min., washed and

air dried.

3.2.4 COUNTING OF ACID.FAST BACILLI: After Ziehl-Neelson staining, the slide

was examined under 1 OOx oil immersion lens, 10 microscopic fields were scanned

in each ring for calculating the number of bacteria per ml of the suspension.

Bacterial ml = ( n x ax D) I ( N x A x V )

where, n= Total number of bacilli counted N= Total number of fields counted

a= Area of spot ( diameter 11mm )

A= Area of microscopic field ( diameter 0.155mm)

D= Dilution

V= Volume of the smear ( 0.001ml)

3.2.5 VIABLE COUNTING OF MYCOBACTERIUM TUBERCULOSIS: Viable bacterial

count was determined to estimate the number of live organisms in a given

suspension. Log dilutions of the bacterial suspension were inoculated on 7H11

plates and incubated at 37°C .. After 4 weeks, the colony forming units (CFU) were

counted and the probable number of mycobacterialml was calculated by multiplying the colony count by the reciprocal of the dilution and the volume inoculated.

3.2.6 PREPARATION OF HEAT KILLED MYCOBACTERIUM TUBERCULOSIS:

Mycobacteria were heat killed by incubating at 80°C for 2 hours (Yang et al,

1995). The heat killed bacilli were inoculated on 7H 11 plates and the absence of

any colony forming units after 4 weeks of incubation confmned the non viability of the culture.

34

Materials and Methods

3.3 CELL CULTURE MEDIA AND REAGENTS

RPMI 1640 (Cat. No. 31800-022), L-Glutamine (Cat.No.21051) and Trypsin

powder (Cat.No.27250) was purchased. from Gibco-BRL, Life Technologies,

USA. EDTA was obtained from Sigma Chemical Co. USA. Fetal Calf Serum

(Cat.No.04-121-1A) was purchased from Biological Industries, Israel. Brewer

Thioglycollate broth was from Difco Laboratories, Detroit, USA. Penicillin was

supplied by Hindustan Antibiotics Ltd, India, Streptomycin from Sarabhai, In~ia

and gentamycin from Fulford Ltd., India.

3.3.1 PREPARATION OF RPMI 1640 MEDIUM: RPMI 1640 supplied as dry

powder by Gibco-BRL was dissolved in 1 liter glass distilled water and

supplemented with 2g NaHC03 and 0.3g L-Glutamine. The medium was sterilized

by filtering through a 0.22J.1 millipore membrane. The sterility was checked by

incubating the medium at 37°C for 48hrs. Penicillin, Streptomycin and

Gentamycin were added to the medium at a concentration of 100U/ml, lOOJ..Lg/ml

and 50J..Lg/ml respectively and the medium was thereafter stored at 4°C.

3.3.2 PREPARATION OF THIOGLYCOLLATE BROTH: Thioglycollate broth used for

the purpose of collection of peritoneal macrophages was prepared as a 4%

solution in glass distilled water. It was sterilized by autoclaving and was aliquoted

in foil covered vials. It was used for intraperitoneal injections in mice after 3

weeks of preparation.

3.4 MAINTENANCE AND CULTURE OF PRIMARY CELLS AND CELL LINES

3.4.1 ELICITED PERITONEAL MACROPHAGES: Adult inbred BALB/c mice were

given an intraperitoneal (i.p) injection of lml of 4% thioglycollate broth. 4-5 days

later the peritoneal cavity was lavaged with 10 ml of ice cold RPMI 164,0. The

cells were spun at 1600rpm and any RBCs in the pellet were lysed by incubating

the resuspended pellet in 0.9% NH4Cl solution for 5min. at 37°C. The cells were

washed twice in warm RPMI 1640 and fmally resuspended in RPMI+10% FCS.

The cells were allowed to adhere to tissue culture treated petri plate for 1hr at 37°

C and 5% C02. After lhr the non adherent population was removed and the

adherent macrophages were replenished with fresh medium. Resident peritoneal

macrophages were collected in a similar manner but without administering

thioglycollate injection.

35

Materials and Methods

3.4.2 NYLON WOOL ENRICHED T CELLS: Spleen cells from six week old BALB/c

mice were prepared in serum free RPMI as reported earlier by Singh et a/, I992. Briefly, the spleens were surgically removed and were triturated in warm medium

containing 2% FCS. The resultant debris was removed by passing the suspension

through a wire mesh. The cells were then passed repeatedly through a 21 G needle to obtain a single cell suspension. The cells were collected by centrifuging at

1800rpm for IOmin. The pellet was incubated for 5min. in lOml of a 0.9% NH4Cl

solution, for lysing the RBC's. The cells were washed x3 times in incomplete

RPMI and a single cell suspension was made by passing the cells through a 2I G

needle. Nylon wool enriched T cells were prepared by the method of Julius et al,

I973. Lymphocytes were enriched by incubating splenocytes for 45min at 37°C in

a plastic petri plate to remove macrophages by plastic adherence. I os cells were

resuspended in 2ml medium and were loaded on a nylon wool column, which had

been previously washed with plain medium, followed by medium with I 0% FCS

and incubated at 37°C for 1 hr. The column was again incubated at 37°C for

45min after loading the cells, and subsequently washed with I5ml of warm

medium. The T cell enriched population was collected as the unbound fraction that

eluted on washing the column.

3.4.3 ENDOTHELIAL CELLS: Endothelial cell line, established from polyoma virus

induced hemangiomas in the skin (s.End) of mice, used in this study was a kind

gift of Dr. Dietmar Vestweber, Max Plank Institute, Frieburg, Germany. These

cells have been characterized and shown to be similar to primary endothelial cells

in terms of adhesion molecule expression, production of chemotactic activity, cell adhesion and capillary tube formation (Bussolino eta/, I99I; Hahne eta/, 1993).

Other studies have validated hemangioma endothelial cell lines as useful models

for studying endothelial cell biology as they possess key features of primary endothelium, including membrane bound ACE (angiotensin converting enzyme),

receptor for acetylated LDL (low density lipoproteins), presence of Von

Willebrand antigen, and capillary tube formation in collagen and fibronectin matrices (Williams et al, I989; Bussolino et a/, 199I and Obeso et a/, 1990).

These cells could therefore, be used as representative of primary endothelial cells, particularly in terms of adhesion molecule function and expression. The cells were cultured and passaged in RPMI I640 with IO% FCS in 75cm2 tissue culture flasks

at 37°C in a 5% C02 incubator .. The cells were dislodged by using a solution of

0.05% Trypsin, 0.53mM EDTA and washed x2 times with RPMI + IO% FCS.

36

Materials and Methods

The viability of the cells was reaffirmed by Trypan blue staining and the cells

were reseeded into tissue culture flasks.

3.4.4 L929 CELLS: The murine fibroblast cell line L929 was obtained from

National Center for Cell Science, Pune, India and was maintained in tissue culture

flasks in RPMI + 10% FCS. The adherent cells were routinely subcultured by

adding a solution of 0.05% Trypsin containing 0.53mM EDTA and incubating for

5min. in a C02 incubator at 3 7° C, in order to dislodge the cells. The cells were

collected in RPMI+ 10% FCS and centrifuged at 1800rpm. The cells were

resuspended in warm complete medium and reseeded.

3.4.5 WEHI 264.1: The murine monocyte-macrophage cell line Wehi 264.1 was

obtained from National Center for Cell Science, Pune, India. It was maintained in

RPMI 1640 supplemented with 5% FCS, the loosely adherent cell population was

harvested by flushing repeatedly with plain medium, followed by centrifuging the

cells prior to reseeding.

3.5 ANTIBODIES, CONJUGATES, RECOMBINANT CYTOKINES AND ELISA KITS

3.5.1 MONOCLONAL ANTIBODIES: All the antibodies used in flow cytometric

analysis were purchased from Pharmingen, USA. These include MAb. to LF A-1 a chain (Cat.No. 01840D), Mac-1 a chain (Cat.No.01710D), Integrin J3 2 chain

(Cat.No.Ol850D), Integrin J3 1 chain (Cat.No.2630D), Integrin a4 chain (Cat.No.

01270D), ICAM-1 (Cat.No. 01540D) and VCAM-1 (Cat.No.Ol810D). Goat

polyclonal antibody to NFKB (Cat.No. sc-372-G) was purchased from Santa Cruz

Biotechnology, California. Monoclonal Hamster anti-murine TNFa (Cat.No.l221-

00), Polyclonal anti-murine TNFa (Cat.No.IP-400), monoclonal hamster anti­

mouse IL-Ia (Cat.No.l837-0l) and polyclonal rabbit anti-mouse IL-IJ3

(Cat.No.80-3688-0l) were all obtained from Genzyme, Cambridge, MA.

3.5.2 CONJUGATES: -Rabbit anti-rat Ig conjugated to FITC (Cat.No.F0234) was

purchased from Dakopatts, Denmark. FITC conjugated Rabbit anti Syrian hamster

IgG was supplied by Jackson lmmunoResearch Laboratories, West Grove, P A.

Horse Radish Peroxidase (HRP) conjugated Rabbit anti Goat Ig was supplied by the Reagent Bank, Nil, India.

3.5.3 CYTOKINES: Recombinant. mouse IL-4 (Cat.No.MIL-4-C),mouse IFNy (Cat.No. MG-IFN) and mouse TNFa (Cat.No.TNF-M) were obtained from

37

Materials and Methods

Genzyme, Cambridge,MA. ELISA kits for mouse IL-Ia (Cat.No.1900-0l), IL-l~

(Cat.No.80-3384-01) and TNFa (Cat.No.80-2802-00) were also supplied by

Genzyme.

3.6 REAGENTS AND INHIBITORS

Sulfanilamide (Cat.No. S9251), n-Naphthyl ethylenediamine dihydrochloride(NEDD) (Cat.No. N9125), NO-methyl L-Arginine (NMMA)

(Cat.No. M7033), sodium nitroprusside(SNP) (Cat.No. S0501),

Pyrrolidinedithiocarbamate(PDTC) (Cat.No. P8765), Lipopolysaccaride(LPS)

(Cat.No; L4391), Sodium Nitrite (S2252), Cycloheximide (Cat.No.C7698) and

Cytochalasin D (Cat.No. C8273) were purchased from Sigma Chemical Co.,

St:..Louis, USA. Tosyl L-lysine chloromethylketone(TLCK) (Cat.No. 874493),

Actinomycin D (Cat.No.102 008), Tyrphostin 47 (Cat.No. 1529 315), Piceatannol (Cat.No. 1500 759), Protein kinase C inhibitor (Cat.No. 1559 648) and Protein

kinase C peptide(l9-31) (Cat.No. 1443 976) were bought from Boehringer Mannheim, W.Germany. Radioactive chromium (51Cr) (Cat.No. NEZ-030S) was

supplied by NEN Research Products, Boston, USA.

3. 7 CHEMICALS

Acrylamide, Bis-acrylamide, ammonium persulphate, N,N,N',N'

tetramethylethylenediamine and ~ mercaptoethanol were purchased from BioRad Laboratories, Richmond, California, USA. Bovine serum albumin, Diaminobenzidine (DAB), Phenyl methyl sulphonyl fluoride, Ethylene diamine

tetra acetate(EDTA), Trizma base, glycine, sodium dodecyl sulphate, Coomassie

brilliant blue G250, Ponceau S, MTT, Dimethylformamide, bromophenol blue,

sodium azide and NP-40 were supplied by Sigma Chemical Co., St. Louis, USA.

Mid range protein molecular weight markers were obtained from Promega,

Madison, USA. Nitrocellulose membranes were supplied by Advanced Microdevices(Pvt.) Ltd., Ambala Cantt., India. Triton X-100, Tween-20, Sodium chloride, Sodium carbonate, Sodium bicarbonate, Disodium orthophosphate, Sodium dihydrogen orthophosphate, Methanol, Acetic acid, Sodium hydroxide and Hydrogen peroxide were all supplied by E. Merck(India) Ltd., Bombay, India. Carbo! fuchsin and methylene blue were from Qualigens Fine Chemicals Ltd., Bombay, India.

38

Materials and Methods

3.8 STANDARDIZATION OF MACROPHAGE INFECTION WITH MYCOBACTERIUM

TUBERCULOSIS

Resident or thioglycollate elicited macrophages were counted and plated on glass

coverslips. The cells were allowed to adhere for 6hrs. and then infected with a

single cell suspension of Mycobacterium tuberculosis diluted in RPMI at different

multiplicities of infection (MOl), and incubated at 37°C in a C02 incubator. After

2hrs. the extracellular bacteria were washed off and the cells were fed with fresh

medium and incubated for a further period of 24hrs. The coverslips were then stained with Trypan blue to assess cell viability or alternatively stained for acid

fast bacilli using carbol fuchsin. The effect of Cytochalasin D on the phagocytosis and internalization of bacteria was assessed by carrying out the infection in presence of this reagent. The cells were treated with Cytochalasin D (0.1-1 OJ..Lg/ml)

2hrs before infection

3.9 FLOW CYTOMETRIC ANALYSIS OF ADHESION MOLECULES EXPRESSED ON

MACRO PHAGES

The expression of cell surface adhesion molecules on macrophages was

determined by Indirect immunofluorescence staining. The cells were plated at a

density of 106 cells /well in a · 6 well plate and treated with the appropriate stimulus. The cells were harvested by flushing with cold wash buffer (PBS

containing 1% FCS and 0.1% sodium azide). Mter washing twice in the wash

buffer, the cells were plated at a density of 1 o6 cells /well in a 96 well round bottom plate. The cells were incubated for 30 min. in PBS containing 5% normal

mouse serum to block the F c receptors. The buffer was discarded and the cells

were then incubated with lOOJ.ll of appropriate monoclonal antibody (l~g/106

cells), diluted in cold wash buffer, and incubated on ice for 45min. After the

incubation, they were washed twice with the wash buffer before incubating with

100J..Ll ofFITC labelled secondary antibody for 45min on ice. The cells were again

washed twice before resuspending in 150J..Ll of 0.4% paraformaldehyde-PBS solution. The fixed cells were analyzed on Bryte (Bio-Rad, USA) flow cytometer. The results obtained were analyzed using the Winmdi program.

3.10 TREATMENT OF MACROPHAGES WITH THE CYTOKINES, IFNy AND IL-4

Macrophage monolayers were stimulated with IFNy (1-IOOU/ml) or IL-4 (1-100ng/ml) for 24hrs and the modulation in expression of adhesion molecules such as ICAM-1, VCAM-1, LFA-1, VLA-4 and Mac-1 was studied by using indirect immunofluorescence staining.

39

Materials and Methods

3.11 INFECTION OF PRIMARY MACROPHAGES WITH MYCOBACTERIUM

TUBERCULOSIS

Macrophages were infected with M tuberculosis at a multiplicity of infection of

10:1 and the change in expression of adhesion molecules was studied, at various

time intervals after infection by using indirect immunofluorescence· staining. To

study the role of phagocytosis in causing modulation in adhesion molecule

expression, the infection and subsequent incubation was carried out in presence of

1 Jlg/ml Cytochalasin D.

3.12 EFFECTOFINFECTION WITH LIVE OR HEAT KILLED AVIRULENT/VIRULENT

STRAIN OF M. TUBERCULOSIS IN CAUSING MODULATION IN EXPRESSION OF

ADHESION MOLECULE ON MACROPHAGES

Macrophages were infected with either live or heat killed mycobacteria of strain

H37Ra (avirulent) or H37Rv (virulent) at a MOl of 10:1. The cells were incubated

for 24 hrs. a~ 37°C in a C02 incubator and then studied for changes in adhesion

molecule expression.

3.13 INFECTION OF MACROPHAGES WITH M. TUBERCULOSIS H37Ra IN THE

PRESENCE OF THE PROTEIN SYNTHESIS INHIBITOR, CYCLOHEXIMIDE

To study the requirement of new protein synthesis by M tuberculosis infected

macrophages, in exhibiting modulation of adhesion molecule expression, the

protein synthesis inhibitor cycloheximide was used. Macrophages were infected

with live M tuberculosis in the . presence of cycloheximide (0.1-1 OOJ.tg/ml) and

fluorescence staining was carried out after 24 hrs. of incubation.

3.14 STUDY OF PROINFLAMMATORY CYTOKINES RELEASED BY MACROPHAGES

INFECTED WITH M. TUBERCULOSIS H37Ra

Macrophage monolayers were established in a 6 well plate and were infected with

Mtuberculosis at a MOl of 10:1. The kinetics of release oflL-1a, lL-1f3 and TNF

a were studied over a period of 24hrs. As a positive control macrophages were

stimulated with 1J.tg/ml ofLPS. Culture supernatant was collected from 3 wells for

each time point, filter sterilized and stored at -70°C. The concentration of · cytokines in the supernatant was determined by sandwich ELISA using Genzyme kits.

40

Materials and Methods

3.15 TNFaBIOASSAY

TNFa bioactivity was measured by utilizing the L929 fibroblast assay as described

by Baarsch et al, 1991. Briefly, L929 cells at a density of 3xi04 cells per well of a

96 well flat bottom plate were incubated overnight in RPMI 1640 supplemented

with 10% FCS to establish a monolayer. Serial 1:4 dilutions of each culture

supernatant to be tested for TNF a bioactivity were prepared in complete RPMI

containing 2.5J..lg/ml of Actinomycin D. Media was removed from the 96 well plate

containing L929 cells, and lOOJ..Ll aliquots of the various dilutions of test samples

were transferred to duplicate wells, and the plate was incubated at 37°C in a C02 incubator. After 18hrs of incubation, 20J..Ll of MTT solution (5mg/ml in PBS) was

added to each well and the plate was incubated for another 3 hrs. After 3 hrs.,

. supernatant were discarded from the wells and the formazan crystals were

dissolved in 1 OOJ..Lllwell of 50% dimethylformamide, 20% SDS solution (pH 4. 7).

The plate was placed on a rocker for 1 hr. after which the color development was

quantitated at a wavelength of 570nm using an automated microplate reader.

Recombinant TNFa was used as a positive control for cytotoxicity. Percent

cytotoxicity was determined from the mean of duplicate wells as follows-

1- OD of cells in test wells X 100

OD of cells in control wells

TNFa units were defmed as the reciprocal of the dilution required to produce 50%

cytotoxicity.

3.16 NEUTRALIZATION OF SELECT CYTOKINES PRODUCED BY MACROPHAGES

INFECTED WITH M. TUBERCULOSIS

Macrophage monolayers were infected with M tuberculosis in the presence of

antibody to IL-l a, IL-l J3 or TNF a. The potential of each antibody in neutralizing

the respective cytokine produced in culture supernatant of infected macrophage

was assessed by using an ELISA. An indirect immunofluorescence staining was

carried out on macrophages after 24hrs. of infection, to study the effect of each cytokine neutralization on modulation of adhesion molecule expression.

3.17 NITRITE MEASUREMENT, AN INDICATOR OF NITRIC OXIDE PRODUCTION

Macrophages were infected with M tuberculosis at different MOl and nitric oxide production, in terms of nitrite accumulation in the culture supernatant, was assessed after 24hrs. by using Griess Reagent as described by Hibbs et al. Briefly,

1 OOJ..Ll of culture supernatant was mixed with an equal volume of Griess Reagent (1% sulphanilamide,0.1% NEDD in 2.5% H3P04) and incubated at room

41

Materials and Methods

temperature for 10 mm. The absorbance was measured at 540mn usmg an

automated microplate reader. Nitrite concentration was calculated from a sodium

nitrite standard curve. All samples were analyzed in triplicates. The effect of

NMMA, an inhibitor of nitric oxide production and SNP, a nitric oxide donor, on

the endogenous production of NO by M tuberculosis infected macrophages was

also studied.

3.18 TREATMENT OF INFECTED MACROPHAGES WITH THE NITRIC OXIDE

INHIBITOR, NMMA OR THE NITRIC OXIDE DONOR, SNP

Macrophages were infected with M tuberculosis in the presence of NMMA ( 10-

1000J..LM) or SNP (1-100J..LM). 24hrs after infection, the modulation of adhesion

molecule expression on the infected macrophages was studied using

immunofluorescence and flow cytometry.

3.19 TREATMENT OF ENDOTHELIAL CELLS WITH THE CYTOKINES, IFNy AND

IL-4

Endothelial cells were plated at a density of 1x106 cells/well of a 6 well plate.

After allowing the cells to adhere for 8 hrs., they were treated with IFNy (1-

lOOU/ml) or IL-4 (0.1-100ng/ml), and incubated at 37°C in a C02 incubator.

After 12hrs. of incubation the cells were trypsinised, washed in complete RPMI

and transferred to a 96 well round bottom plate. The cells were washed again in

PBS-azide-FCS buffer and resuspended in 100J.!l of the appropriate antibody

diluted in the wash buffer. The cells were incubated for 45 min on ice and then

washed twice in the wash buffer. After this, the cells were incubated with the

relevant secondary antibody conjugated to FITC and again incubated on ice for 45

min., in the dark. After the incubation period, the cells were washed twice, and

resuspended in 150J..Ll of 0.4% paraformaldehyde in PBS. The cells were analyzed

on the Bryte (Bio-Rad, USA) flow cytometer. The results obtained were analyzed

using the Wimndi program.

3.20 TREATMENT OF ENDOTHELIAL CELLS WITH CULTURE SUPERNATANT OF

M. TUBERCULOSIS INFECTED MACRO PHAGES (CONDITIONED MEDIUM)

Macrophage monolayers were infected with M tuberculosis at a MOl of 10:1 and

culture supernatant was collected from them at different time intervals after

infection and filter sterilized. Endothelial cells were stimulated with the

conditioned medium and incubated at 37°C in a C02 incubator for 24 hrs. The

·cells were subsequently stained by an indirect immunofluoresence method to study

42

Materials and Methods

the modulation in adhesion molecule expression. In another set of experiments,

endothelial cells were treated with culture supernatant from M tuberculosis

infected macrophages and incubated for different time intervals after stimulation.

Immunofluoresence staining and flow cytometric analysis was carried out to study

the time kinetics of change in the expression of adhesion molecules on endothelial cells.

3.21 TREATMENT OF ENDOTHELIALCELLS WITH CONDITIONED MEDIUM IN

THE PRESENCE OF PROTEIN SYNTHESIS INHIBITOR, CYCLOHEXIMIDE

To study the requirement of new protein synthesis by endothelial cells, for exhibiting modulation of adhesion molecule expression, the protein synthesis inhibitor cycloheximide was used. Endothelial cells were treated with conditioned

medium in the presence of cycloheximide (lOJ..Lg/ml) and fluorescence staining was

carried out after 12hrs. of incubation.

3.22 EFFECT OF CYTOKINE NEUTRALIZATION ON THE CHANGE IN EXPRESSION

OF ADHESION MOLECULES ON ENDOTHELIAL CELLS INDUCED BY CONDITIONED

MEDIUM

Culture supernatant was collected from M tuberculosis infected macrophages 24

hrs. after infection and aliquoted. Different aliquots were treated with antibody to

either IL-Ia, IL-lf3 or TNFa and incubated for 2 hrs at 37°C. The concentration

of antibody to be used was decided on the basis of the amounts of each cytokine

present in the supernatant. Endothelial cells were then treated with culture supernatants that had been neutralized for a select cytokine, and subsequently stained and analyzed on the flow cytometer.

3.23 TREATMENT OF ENDOTHELIAL CELLS WITH CONDITIONED MEDIUM IN

THE PRESENCE OF INHIBITORS OF NFKB ACTIVATION

Endothelial cells were treated for 2 hrs. with the antioxidant, PDTC {lJ..LM-lmM)

or the serine protease inhibitor, TLCK(lJ..LM-lmM) both of which are known

inhibitors ofNFKB activation. The cells were then stimulated with the conditioned medium, in the presence of these reagents, incubated for a further 12 hrs. and subsequently immunostained and analysed by flow cytometry.

43

Materials and Method:s

3.24 IMMUNOBLOT DETECTION OF THE NFKB 65KDA SUBUNIT IN THE

CYTOPLASMIC AND NUCLEAR FRACTIONS OF STIMULATED OR UNSTIMULATED

ENDOTHELIAL CELLS

3.24.1 Preparation of cytoplasmic and nuclear extracts: Endothelial cells were

either left unstimulated or were stimulated with culture supernatant from M

tuberculosis infected macrophages in the presence or absence of 1 OOJ..!M PDT C. Cytoplasmic and nuclear extracts were prepared from these cells ( 6x 106 cells for each sample) 15, 30, 45 and 60 min. after stimulation, according to the method of

Dignam et a/, 1983. All extractions were performed on ice with ice cold reagents.

Briefly, cells were washed twice with PBS and harvested by pelleting at 3000rpm.

The pellet was resuspended in three packed cell volumes of the Hypotonic buffer (IOmM HEPES, 1.5mM MgCI2, lOmM KCl, 0.2% NP-40, 0.2mM PMSF, 0.2mM

DTT, pH 7.9 at 4°C) and incubated on ice for 10 min. The lysate was spun at

3300g for 15min. and the supernatant was collected and stored as the cytoplasmic fraction. The pellet, which contained the nuclei, was resuspended in half packed nuclear volume of the Low salt buffer (20mM HEPES, 10% glycerol, 1.5mM

MgC12, 20mM KCl, 0.2mM EDTA, 0.2mM PMSF, 0.5mM DTT, pH 7.9). To this

was added half packed nuclear volume of High salt buffer (Low salt buffer with 1.2M KCl) in a dropwise manner with gentle stirring. The mixture was incubated

on ice for 30min with occasional vortexing. Nuclear proteins were isolated by

centrifugation at 25000g for 30min. Protein concentration was determined by Bradford assay and stored at -70°C until use for SDS PAGE.

3.24.2 SDS PAGE of cytoplasmic and nuclear fractions: Equal concentrations

of nuclear and cytoplasmic fractions of each sample were boiled in equal volume

of Loading buffer (125mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol, 10% J3

mercaptoehanol) and resolved on 10% SDS polyacrylamide gel at 15mA in a

Minigel electrophoresis system (Hoefer Scientific Instruments, San Francisco, CA) according to the discontinuous buffer system of Laemmli, 1970. The resolved

components were electrophoretically transferred to a nitrocellulose membrane by the me9tod of Towbin eta/, 1979 in a Bio Rad Trans Blot apparatus using Iris­Glycine buffer (pH 8.3) at 30mA overnight.

3.24.3 Western blot analysis: The nitrocellulose membrane was blocked with a solution of 5% dried milk in PBS-0.05% Tween 20 for 1 hr at room temperature. The blot was rinsed once with PBS and then incubated for 1hr at room temperature with the goat polyclonal antibody to the 65kDa subunit ofNFKB (20J..Lg/ml in PBS­Tween 20). After washing thrice with PBS-Tween, the blot was incubated for 1 hr at room temperature with I: 100 dilution of Rabbit a Goat Ig conjugated to HRP.

44

Materials and Methods

The blot was washed thrice with PBS-Tween and then developed with 0.5mglml

DAB in PBS containing 0.002% H202. The substrate was made fresh.

3.25 TREATMENT OF ENDOTHELIAL CELLS WITH CONDITIONED MEDIUM IN

THE PRESENCE OF INHIBITORS OF SIGNAL TRANSDUCTION PATHWAYS

Endothelial ce~ls were treated for 2 hrs. with one of the following signal

transduction inhibitors-

Tyrphostin 47 (lOOpM-hnM), an inhibitor of Protein Tyrosine Kinase

Bisindolylmaleimide (0.1nM-1 J.tM), an inhibitor of Protein Kinase C

The cells were subsequently stimulated with the conditioned medium, in the

presence of these inhibitors and incubated at 37°C for various time intervals.

Immunofluoresence staining was then carried out on the cells following which

they were analysed on a flow cytometer.

3.26 STANDARDIZATION OF ADHESION ASSAYS AND BLOCKING STUDIES

Endothelial cells were grown as monolayers on 12mm coverslips in 24 well plates.

The cells were cultured in RPMI 1640+ 10% FCS. Nylon wool enriched T cells

were harvested from flasks and were resuspended in 1 ml medium containing 2.5%

FCS at the density of 107 cells/ml. The cells were labeled with 51Cr at

approximately 300-350J.tCiii07 and incubated for 1 hr at 37°C in a C02 incubator

with frequent shaking. After labeling, the cells were washed three times with

medium and fmally resuspended in RPMI+5% FCS. Chromium labeled T cells

were added to the endothelial cell monolayers and allowed to adhere for an

appropriate time at 37°C in a C02 incubator. The non adherent cells were removed

byr gently rinsing the coverslips in warm medium. The remaining adherent cells

were lysed with of 0.2% NP-40 and the counts were taken for triplicate samples in

a Gamma counter (LKB, Wallace,USA). The percent adherence was calculated

relative to the control as follows,

% Adhesion = Average count in sample well x 100

Average count in control well

Endothelial cells were treated with IFNy (1-100U/ml) or IL-4 (0.1-lOOng/ml) for 12 hrs. and the adherence of T cells to such a stimulated monolayer was studied to answer the question whether an increase in the expression of adhesion molecules

on the endothelial cells would correlate with an enhanced adhesion ofT cells to it. Experiments were also · carried out to ascertain the specificity of adhesion by

fmding out whether antibodies to adhesion molecules could significantly block the

45

Materials and Methods

cytokine induced adhesion. Antibodies to the Ig superfamily molecules were added to the cytokine activated endothelial cells 30min. before the adhesion assay. Mter

the 30min. incubation the antibodies were washed off and the assay was performed

as described. For assaying the adhesion molecule usage of leukocytes, antibodies to various

integrin molecules were coincubated with the chromium labeled cells for 30min.

The cells were washed once and then allowed to adhere to activated or unactivated

endothelial cells. The adhesion was assayed as described.

3.27 ADHESION OF T. CELLS TO ENDOTHELIAL CELLS STIMULATED WITH

·cuLTURE SUPERNATANT OF M. TUBERCULOSIS INFECTED MACROPHAGES

Confluent monolayers of endothelial cells grown on coverslips were treated with

culture supernatant of M tuberculosis infected macrophages and incubated for 12

hrs. at 37°C in a C02 incubator. Nylon wool enriched T cell was labeled with 51Cr

at 300-350J.1Cill07 cells and incubated for 1 hr at 37°C. After labeling, the cells

were washed thrice with warm medium and fmally resuspended in RPMI+5% FCS at a cell density of 5 x 1 o6 cells/mi. 5 x 1 os labeled T cells were added per well to

the endothelial monolayer and incubated at 37°C for 45 min. After the incubation, the non adherent population was removed by rinsing the coverslips 10 times in

warm medium. The remaining adherent cells were lysed with 200Jll of 0.2% NP-

40 solution in RPMI, counts were taken in a Gamma counter and the percent adhesion was calculated. In some experiments, stimulated endothelial cells were

treated with antibodies to ICAM-1 or/and VCAM-1 for 30 min before the assay.

After the incubation, the antibodies were washed off and the assay was performed. For assaying the integrins used by T cells for adhering to the stimulated

endothelial monolayer, labeled T cells were treated for 30 min. with antibody to

LFA-1, VLA-4, Mac-1, PI or P2 integrins. The cells were washed once and then used for the assay as described.

3.28 ADHESION OF WEHI 264.1 CELLS TO ENDOTHELIAL CELLS STIMULATED

WITH CULTURE SUPERNATANT OF M. TUBERCULOSIS INFECTED MACROPHAGES

Endothelial cells were grown on coverslips and were treated with culture supernatant of M tuberculosis infected macrophages for 12 hrs. The monocyte macrophage cell line Wehi 264.1 was labeled with 51Cr (300-350J.1Cii107 cells)

and incubated for 1 hr.at 37°C with frequent shaking. After labeling, the cells were

washed thrice with warm medium and resuspended in RPMI at a cell density of

46

Materials and Methods

2.5x106 cells/mi. The labeled cells were incubated for 30 min. at 37°C with 5%

heat inactivated normal mouse serum to block the Fe receptors. The labeled Wehi

cells were added to the endothelial monolayer at a concentration of 2.5x105

cells/well and incubated at 37°C for 30 min. After the incubation, the non adherent

population was removed by rinsing the coverslips 15 times in warm medium. The

remaining adherent cells were lysed with 200J.ll of 0.2% NP-40 solution in RPMI,

counts were taken in a Gamma counter and the percent adhesion was calculated. In

some experiments, stimulated endothelial cells were treated with antibodies to

ICAM-1 or/and VCAM-1 for 30 min before the assay. After the incubation, the

antibodies were washed off and the assay was performed. For assaying the integrins used by W ehi 264. 1 for adhering to the stimulated endothelial

monolayer, the labeled cells were treated for 30 min. with antibody to LFA-1,

VLA-4, Mac-1, f3 1 or P2 integrins. The cells were washed once and then used for

the assay as· described.

3.29 IN VIVO STUDY TO TEST THE POTENTIAL OF CULTURE SUPERNATANT

FROM M. TUBERCULOSIS INFECTED MACROPHAGES IN INDUCING CELL

INFILTRATION

Macrophage were plated in 6 well plates at a cell density of 1 o6 cells/well and

infected with M tuberculosis at a MOl of 10:1 in a fmal volume of 250J.!l per well.

The culture supernatant was collected 12 hours after infection and was used to

inject naive BALB/c mice sub-cutaneously in the thigh region. Mice that were

injected with ~ulture supernatant of uninfected macrophages and processed

similarly served as a control. In some experiments mice were injected with culture

supernatant that had been neutralised for TNFa by incubating the culture

supernatant for 2 hrs at 37°C with an antibody to TNFa. In another series of

experiments, mice were injected intravenously with 50J.lg each of antibody to the

Ig superfamily molecules ICAM-l and VCAM-1 or the integrins LFA-1, Mac-1

and VLA-4, to assess the contribution of these adhesion molecules in causing cell

infiltration. The site of injection was marked carefully and was removed 12 or 24

hours post injection after sacrificing the mouse. The excised tissues were fixed in

10% formalin-PBS, embedded in paraffin wax and sections of 5J.lffi thickness were

obtained and were stained using Haematoxylin and Eosin dyes. Alternatively, the

tissue was immediately cryofixed and was stored at -70°C. The frozen tissue was

embedded in OCT compound and cryosections of 5 J.lffi thickness were cut in a

cryostat, at a temperature of 17-19°C. The sections were dried overnight at room

47

Materials and Methods

temperature, and fixed by immersing in acetone for 10 minutes the next morning.

The sections were immediately stored at -70°C.

3.30 IMMUNOFLUORESCENT STAINING OF ICAM-1 AND VCAM-1

MOLECULES ON TISSUE SECTIONS

For immunohistochemical staining, the sections were rehydrated by immersing for

10 minutes in a solution of 50mM Tris/150 mM NaCl (pH 7.2). Thereafter, the sections were incubated for 45 minutes in a humidified chamber, with rat anti

mouse ICAM-1 or VCAM-1 at a concentration of 10~g/ml. Purified rat lgG used

at 10~glml was included as a control. Mter incubation with the primary antibody,

the sections were washed thrice for two minutes each in the Tris/NaCl solution.

Next, they were incubated for 45 minutes with rabbit anti rat IgG*FITC at a·

dilution of 1:50. After the final washes in Tris/NaCl, the sections were mounted in a solution of glycerol-PBS to which a crystal of pPD had been added. The mounted sections were visualized under UV illumination and photographs were

taken on a Microphot FX microscope system (Nikon, Japan).

48