dynamic changes in neutrophil defensins during endotoxemia · vancouver, british columbia, canada...

INFECTION AND IMMUNITY,0019-9567/01/$04.00�0 DOI: 10.1128/IAI.69.12.7793–7799.2001

Dec. 2001, p. 7793–7799 Vol. 69, No. 12

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Dynamic Changes in Neutrophil Defensins during EndotoxemiaM. E. KLUT,* B. A. WHALEN, AND J. C. HOGG

University of British Columbia McDonald Research Laboratories, iCAPTURE Centre, St. Paul’s Hospital,Vancouver, British Columbia, Canada

Received 20 June 2001/Returned for modification 8 August 2001/Accepted 18 September 2001

Bacterial endotoxin or lipopolysaccharide (LPS) is an important causative agent of sepsis. This studydetermines the expression of defensins NP-2 and NP-5 and the function of polymorphonuclear leukocytes(PMN) in rabbits treated with LPS. PMN functional activity was assessed by measuring CD18 expression andH2O2 production and by examining the lungs. NP-2 and, to a minor extent, NP-5 of circulating PMN increaseduring endotoxemia. This early increase is concomitant with neutrophilia and elevated CD18 expression andH2O2 production, as well as with enhanced NP-2 immunoreactivity in pulmonary microvessels. A decline indefensins, shortly after the last LPS treatment, is associated with a decrease in the circulating activated PMNand enhanced immunoreactivity in the inflammatory cells, as well as with lung tissue damage. This study showsthat LPS-induced changes in the defensins of circulating PMN correlate with the number and activatedcondition of these cells and suggests that PMN-derived products implement the inflammatory reaction thatleads to lung injury and sepsis.

Endotoxin from gram-negative bacteria causes a number ofpathophysiological effects that can lead to the adult respiratorydistress syndrome (ARDS). Despite the progress made overthe years, ARDS is still a major cause of death among patientswho develop sepsis or pneumonia (17). A major factor thatcontributes to this high mortality is multiple organ failure as-sociated with tissue injury. Lung injury typical of this syndromehas been associated with polymorphonuclear leukocytes(PMN) (12). These short-lived, bone marrow-derived cells arerecruited to inflamed sites by responding to bacterial productsand inflammatory mediators such as interleukins, tumor ne-crosis factor, and complement fragments. During this processPMN play a key role in the body’s defense, but they can alsodamage the tissue that they are attempting to protect (33).Their granules carry a wide range of powerful antimicrobialenzymes and peptides (7, 19). Six of these neutrophil peptides(NP-1, -2, -3A, -3B, -4, and -5) or defensins have been isolatedand characterized in rabbits. Among these, NP-2 of PMN isstructurally and functionally identical to MCP-2 of adult rabbitalveolar macrophages (30). Defensins are known to mediateinflammation (40) and cause damage to endothelial (29), epi-thelial (23, 24), and tumor (20) cells. We have previously shownthat immature marrow PMN contain elevated levels of NP-2 andNP-5 (16), and it has been shown that PMN transit time throughthe marrow decreases with bacterial infection (34, 37). Thepresent study was designed to extend these observations by de-termining the level of defensins and the functional activity ofcirculating PMN during endotoxemia. PMN functional activitywas assessed by measuring the expression of �2 integrin CD18and production of H2O2 and by examining the lungs.

MATERIALS AND METHODS

Experimental protocol. Adult female New Zealand White rabbits (2.2 � 0.2kg) were injected daily for 5 days via the marginal ear vein with either normalsaline or lipopolysaccharide (LPS) from Esherichia coli 055:B5 (Sigma, St. Louis,Mo.). Increasing doses of LPS were given as 10 �g on the first two days, 20 �gon the following two days, and 30 �g on the last day of the experiment. Periph-eral blood (2 ml) was collected at zero time (baseline), 24 h after each injection,and 1 h following the last treatment. Leukocyte cell counts were carried out in anS 880 Coulter Counter (Beckman Coulter, Inc., Miami, Fla.). Cells of PMNlineage were counted on Wright-stained blood smears. Animals were sacrificedwith an excess of ketamine and xylazine at 49 or 97 h. The base of the heart wasligated, and the lungs were excised. The right lung was perfusion fixed with 1%paraformaldehyde (PFA) and inflated with an optimal cutting temperature em-bedding medium (Miles, Elkhart, Ind.) before storage at �70°C. The left lungwas processed for transmission electron microscopy (TEM).

Flow cytometry. (i) Neutrophil peptides NP-2 and NP-5. Peripheral blood cellsuspension (4.0 � 106 cells/ml) from saline (n � 3)- or LPS (n � 5)-treatedrabbits was fixed with 0.8% PFA. Red blood cells (RBCs) were lysed with animmunolysing agent (commercial kit from Beckman Coulter, Inc.). After beingwashed with phosphate-buffered saline, pH 7.3, leukocytes were simultaneouslyfixed and permeabilized (1 h) with 0.7% PFA and 50 �g of L-�-lysophosphati-dylcholine/ml. Cells were incubated (1 h) with 9 �g of mouse monoclonal anti-bodies B9 (anti-NP-2 and anti-MCP-2) or R5-3 (anti-NP-5) (25) or the nonspe-cific mouse immunoglobulin G1 (IgG1) (Sigma) per ml. Cells were then labeled(1 h) with a 1/50 dilution from stock of fluorescein isothiocyanate-conjugatedanti-mouse secondary antibody (Sigma). After fixing with 0.8% PFA, and toexclude cell aggregates, cells were stained (15 min) with 4 �g of propidium iodideper ml (16). The mean fluorescence intensity of 5,000 to 30,000 cells was mea-sured using analysis gates for PMN in a flow cytometer (Epics XL; BeckmanCoulter, Inc.).

(ii) CD18. Blood samples from saline (n � 4)- or LPS (n � 6)-treated rabbitswere collected into tubes containing acid-citrate-dextrose. Aliquots (1 ml) ofthese samples were added to 1.5 ml of Hank’s balanced salt solution, pH 7.3.After being fixed (10 s) with 1.6% PFA, cells were incubated (15 min) with l �gof mouse anti-human LFA-1, �-chain CD18 (Dako, Glostrup, Denmark), ormouse IgG (Sigma) per ml. Samples were incubated (15 min) with 7.5 �g of goatanti-mouse fluorescein isothiocyanate per ml. After lysing of the RBCs, cellswere fixed with 1% PFA and the mean fluorescence intensity of 3,000 cells wasmeasured by flow cytometry.

(iii) H2O2. Blood (2 ml) from saline (n � 3)- or LPS (n � 3)-treated rabbitswas drawn into EDTA-containing tubes. Aliquots (50 �l) of blood cell suspen-sion (3.5 � 10 6 cells/ml) or cellZyme (positive control) were incubated (5 min)at 37 oC, with 25 �l of dichlofluorescein hydrogen diacetate (Cellprobe; CoulterElectronics) or phosphate-buffered saline (control) and then placed on ice. Afterthe cells were stained with 5 �g of propidium iodide/ml, RBCs were lysed and the

* Corresponding author. Mailing address: iCAPTURE Centre, Mc-Donald Research Laboratories, UBC, St. Paul’s Hospital, 1081 Bur-rard St., Vancouver, BC, Canada V6Z 1Y6. Phone: (604) 806-8346.Fax: (604) 806-8351. E-mail: [email protected].

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shift of the peak position from the control to the test was measured by flowcytometry.

(iv) NP-2 (MCP-2) immunoreactivity. The modified version of the alkalinephosphatase anti-alkaline phosphatase method (16) was used to detect NP-2(MCP-2) in the lungs of rabbits treated with LPS or saline. Right lung frozensections (4 �m) were obtained on a cryostat (Frigocut 2800 N; Leica) andmounted on 3-aminopropyltriethoxysilane-coated slides. After blocking of thenonspecific binding with 5% rabbit serum, specimens were labeled (1 h) with 0.5�g of B9 (anti-NP-2 and anti-MCP-2) antibody per ml. Dilutions were made in

0.05 M Tris-buffered saline, pH 7.6, containing 1% bovine serum albumin, andthe nonspecific mouse IgG1 was used as a negative control. After rinsing, spec-imens were incubated (30 min) with a 1:20 dilution of rabbit anti-mouse IgG(Dako). Specific labeling was detected by incubating (30 min) with a 1:50 dilutionof a mouse alkaline phosphatase anti-alkaline phosphatase complex (Dako)followed by a new fuchsin-based red substrate solution. Specimens were coun-terstained with Mayer’s hematoxylin, dehydrated with ethanol, and mounted inEntalan (BDH, Mississauga, Ontario, Canada). Photographs were obtained us-ing 100 ASA Kodak film in a Zeiss light microscope.

FIG. 1. (A and B) Kinetics of NP-2 and NP-5 in circulating PMN. NP-2 increases by 24 to 48 h (P 0.05) (A), and NP-5 rises at 48 h (P �0.19) (B). Both of these peptides fall at 97 h. Values show means � standard errors of the means. P 0.05 from the baseline (�).

TABLE 1. PMN and band forms (as % of total white blood cells)a

Cell type Treatment% Of total white blood cells at:

Baseline 24 h 48 h 72 h 96 h 97 h

PMN Saline 31.8 � 2.9 29.3 � 4.2 23.8 � 0.9 25.5 � 1.6 35.7 � 3.6 34.7 � 5.0LPS 38.0 � 3.1 64.6 � 5.2* 57.6 � 3.4* 49.1 � 4.6 45.4 � 5.2 17.9 � 5.6*

Bands Saline 1.2 � 0.7 0.3 � 0.2 0.2 � 0.2 0.5 � 0.2 1.7 � 0.5 0.7 � 0.2LPS 2.1 � 0.7 6.3 � 1.3* 2.1 � 0.6 1.0 � 0.2 1.0 � 0.6 2.6 � 1.0

a Note that circulating PMN increase at 24 and 48 h and decrease at 97 h of LPS treatment. By comparison, band forms increase only at 24 h. Values represent mean� standard errors of the means. Saline, n � 6, LPS, n � 8. P 0.05 from the baseline (*).

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(v) TEM. The left lung was inflated and immersion fixed (1 h) with 2.5%glutaraldehyde using 0.1 M Na cacodylate, pH 7.3. Lung tissue samples (2mm3) were further fixed (1 h) in 2.5% glutaraldehyde and postfixed (1 h) in 1%osmium tetroxide. Samples were then dehydrated with ethanol and embedded inLRWhite. Ultrathin sections were stained with uranyl acetate and lead citrateand examined on a Philips 400 electron microscope.

(vi) Statistical analysis. One-way analysis of variance and a paired t test wereused for multiple- and two-group comparisons. Data are presented as mean �standard error of the mean, and statistical significance is defined as P 0.05.

RESULTS

Peripheral blood leukocytes. Rabbits received daily injec-tions of LPS or saline for 5 days. Total leukocyte, PMN, andband cell counts were carried out at the baseline (0 h), 24 hfollowing each dose, and 1 h after the last injection (97 h). Weobserved that leukocyte counts increase during LPS treatment.As shown in Table 1, PMN increase at 24 and 48 h and bandforms rise at 24 h (P 0.05). As expected, PMN countsdecrease at 1 h after the last dose of LPS (97 h) (P 0.05) withno change in the band cells. By comparison, cell counts do notchange with saline treatment (P � 0.05).

Flow cytometry (i) NP-2 and NP-5. Figure 1A shows thatNP-2 in circulating PMN increases 1.8-fold at 24 and 48 h ofLPS treatment. A sharp drop (6.9-fold) in this peptide is ob-served following the last dose of LPS (97 h) (P 0.05). Figure1B shows that the amount of NP-5 tends to rise at 48 h (P �0.19) and declines (3.2-fold) at 97 h (P 0.05). The amount ofneither of these peptides changes with saline treatment (P �0.05).

(ii) CD18 and H2O2. Figure 2A shows that the expression ofCD18 on the surface of circulating PMN increases during LPStreatment and reaches a peak (2.2-fold) at 48 h. A sharpdecline in this integrin is observed after the last LPS exposure(97 h compared to 96 h) (P 0.05). Figure 2B shows thatH2O2 production also increases with repeated LPS injections,peaking (2.8-fold) at 48 h (P 0.05) and falling at 97 h (P �0.08). Neither of these markers of PMN activation changesduring saline treatment (P � 0.05).

(iii) NP-2 (MCP-2) immunoreactivity. Light microscopy re-sults show that, at 49 h, the NP-2 immunoreactivity in pulmo-

FIG. 2. (A and B) CD18 expression and H2O2 production in circulating PMN. CD18 expression (A) and H2O2 production (B) increase withrepeated doses of LPS and exhibit a peak at 48 h. These parameters fall at 97 h and do not change with saline treatment. Values represent means �standard errors of the means. P 0.05 from the baseline (�) or from 96 h (��).

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FIG. 3. (A to F) NP-2 (MCP-2) immunoreactivity. PMN-containing defensins are scarce in the lungs of control rabbits (arrows) (A) andaccumulate in the capillaries of animals treated with LPS for 49 h (arrows) (B). Defensin-rich phagocytes are prominent in the interstitial (arrows)(C) and alveolar (D) spaces and are less evident in the capillaries of animals treated with LPS for 97 h (E). (F) IgG1-negative control.Magnifications are as follows: �200 (A, B, E, and F), �700 (C), and �850 (D).


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nary microvessels of random sections from control rabbits ismoderate (Fig. 3A, arrows) compared to that of LPS-treatedanimals (Fig. 3B, arrows). By comparison, at 97 h of LPStreatment, NP-2 (MCP-2)-positive cells appear in the intersti-tium (Fig. 3C, arrows) and alveoli (Fig. 3D) and are less evi-dent in the capillaries (Fig. 3E). IgG-incubated lung sectionsshow no evidence of staining (e.g., specimens from LPS-treated animals shown in Fig. 3F).

(iv) TEM. Ultrastructural examination of random lung sec-tions at 49 h shows that leukocytes, mostly PMN and mono-cytes, form clusters inside microvessels and that some of thesePMN are closely attached to the endothelium (Fig. 4A). At97 h of LPS treatment, endothelial cells display vacuolizationand membrane blebbing (Fig. 4B, arrows). Inflammatory cells

appear in the interstitium and alveoli (Fig. 4C, arrows), and thewalls of the alveolar septa exhibit electron lucent spaces thatsuggest edema. Epithelial type II cells are flattened, extendprotrusions over type I cells, and display reduced microvilli.The alveolar space shows fibrin deposits typical of hyalinemembrane formation (Fig. 4D, arrows).

DISCUSSION

This study shows that E. coli endotoxin (LPS) increases theexpression of defensins NP-2 and NP-5 in circulating PMN.This increase is associated with elevated numbers of seg-mented and nonsegmented PMN. We have previously shownthat immature marrow PMN contain high levels of defensins

FIG. 4. (A to D) Changes in the lungs of rabbits treated with LPS for 49 h (A) and 97 h (B to D). Note PMN adhered to the endothelium (A),endothelial cell vacuolization (B), interstitial cell infiltrates (C), and alveolar epithelial cell damage with fibrillar deposits (D). Magnifications areas follows: �7,700 (A), �2,150 (B), �2,750 (C), and �10,000 (D).


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(16). In the present study, a rise in defensins is likely due tomaturational and/or conformational changes in the peptideprecursors of less mature PMN. Results indicate that thesechanges are not synchronized with the transformation of thenucleus from a band to a multilobed form, which supports thenotion that nuclear morphology is not always concordant withcellular function (18).

Earlier investigations demonstrated that human defensinsare synthesized as large 94-amino-acid (aa) precursors whichmust undergo cleavage to yield 75-aa and 56-aa prodefensins(9, 22, 36). These intermediate forms of defensins are presentin circulating PMN (22) and are pH sensitive in their process-ing to smaller peptides (7, 9). Other studies showed that bac-terial LPS recognizes PMN surface receptors and is internal-ized into phagosomes (15, 26, 27). It is possible that thefollowing incorporation of primary granule contents intophagosomes (3) sets the ideal environment for the proteolyticprocessing that promotes defensin expression and microbialdestruction. In this respect, it is interesting that Chediak-Hi-gashi syndrome patients show reduced transfer of lysosomalenzymes to phagocytic vacuoles and are highly susceptible tomicrobial infections (33).

Since circulating PMN retain considerable ability to synthe-size proteins (3, 22, 33), we cannot rule out the possibility thatincreased amount of defensins could, at least in part, resultfrom de novo biosynthesis. Under normal circumstances, ma-ture PMN have a short life span and contain vast quantities ofgranule proteins; thus, biosynthesis may not be necessary.However, during inflammation, de novo protein synthesis maybe required to sustain functional activity (21) and compensatefor rapid granule turnover (33). It has been previously shownthat LPS triggers the expression of mRNA for �-defensins,which may serve as a protective mechanism to defend the hostagainst invading microorganisms (5, 28).

In the present study, the early (24 to 48 h) rise in PMNdefensins is concomitant with cell activation, increased PMNadherence to the vascular endothelium, and enhanced NP-2immunoreactivity inside microvessels, which creates opportu-nity for cell toxicity via oxidative and nonoxidative mecha-nisms. Defensins are known to increase their cytotoxicity byacting synergistically with hydrogen peroxide (24). The com-bined effect of these PMN-derived products is illustrated inspecific-granule-deficiency patients, who show normal respira-tory burst but are virtually devoid of defensins, or in patientswith chronic granulomatous disease, who express defensins butfail to generate reactive oxygen intermediates and are predis-posed to life-threatening infections (6).

The observed decline in the defensins of circulating PMN at97 h is associated with a drop in the number of activatedcirculating PMN and correlates with PMN exudation from thevascular space as well as endothelial and epithelial cell dam-age. Strong immunoreactivity for defensins in the phagocytesof the interstitium and alveoli is associated with amplifiedinflammatory reaction and is attributed to upregulation of thegene encoding MCP-2 (8) or to phagocytosis of apoptoticPMN.

Furthermore, the weak NP-2 immunoreactivity observed inPMN of pulmonary microvessels suggests extracellular degran-ulation. Extracellular granule release has been previously dem-onstrated during phagocytosis (39) and following cell activa-

tion (2, 6). High concentrations of defensins have beendetected in the plasma and other body fluids of patients withbacterial infections, chronic bronchitis, cystic fibrosis, idio-pathic pulmonary fibrosis, ARDS, chronic obstructive pulmo-nary disease, and �1-antitrypsin deficiency (1, 2, 10, 13, 25, 31,32). Although the in vivo role of high concentration of de-fensins is presently unclear, recent studies showed that thesepeptides mediate lung inflammation and dysfunction (40). Fur-thermore, data from in vitro studies suggest that defensins maycontribute to cytotoxicity by promoting cytokine productionand leukocyte recruitment, inducing mast cell degranulation,decreasing antioxidant levels, or altering the permeability andpotential of the cell membrane (10, 14, 23, 24, 35, 38). Otherstudies indicate that an exacerbation of the latter events maytake place near cholesterol-enriched membranes (4) in thepresence of lipoproteins (11) or in areas of difficult access tonaturally occurring inhibitors, such as �2 macroglobulin and �1

proteinases (6). Since excessive amounts of defensins promoteinflammation and bacterial adhesion (10, 40), their role in thepathogenesis of clinical disorders associated with bacterial in-fection must be further investigated.

In summary this study shows that bacterial endotoxin causeschanges in the defensins and functional activity of PMN andindicates that the excessive turnover of PMN-derived productselicits an amplified inflammatory reaction that is detrimentalto the lung.

ACKNOWLEDGMENTS

This work was funded by Canadian Institutes of Health ResearchCIHR-4219 and the British Columbia Lung Association.

We thank T. Ganz from the University of California at Los AngelesSchool of Medicine for insightful reading of the manuscript and thekind supply of B9 and R5-3 antibodies.

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