role of complement and polymorphonuclear cells in

Role of Complement and PolymorphonuclearCells in Demethylchlortetracycline-inducedPhototoxicity in Guinea Pigs

INHIBITION BY DECOMPLEMENTATIONIN VIVO

HENRYW. LIM, HOWARDNOVOTNY,and IRMA GIGLI, Division of Dermatology,University of California, San Diego, California 92103; Department ofDermatology and the Irvington House, New York University Medical Center,New York 10016

A B S T R A CT In this study, demethylchlortetracy-cline was used as a prototype of exogenous phototoxicsubstarnces. In vitro, exposure of serum containing de-methylchlortetracycline to ultraviolet-A irradiationresulted in the diminution of total complement he-molytic activity and C4, C2, C3, and C5 activities. Inaddition, chemotactic activity for human polymor-phonuclear cells was generated, which was thermosta-ble and antigenically related to human C5 but nothuman C3. In vivo, phototoxic lesions were inducedin guinea pigs upon intradermnal injections of demeth-ylchlortetracycline solution, followed by ultraviolet-Airradiation. On a scale of 0-3+, the animals developeda maximal response of 2.5 at 20 h. This clinical re-sponse was associated with cellular infiltrate in thedermis, consisting of 29±2% of neutrophils at 24 h.The participation of the polymorphonuclear cells wasevaluated in guinea pigs rendered neutropenic bytreatment with cyclophosphamide. In these guineapigs, demethylehlortetracycline and ultraviolet-A in-duced a maximal response of 0.75±0.6, which was as-sociated histologically with 1.2±0.5% neutrophils inthe dermis. The role of complement in this process wasstudied in guinea pigs congenitally deficient in C4, and

This work was presented, in part, at the Annual Meetingof the American Association of Physicians, Washington DC,1982, and published in abstract form: 1982. Clin. Res.20:552a. (Abstr.)

Address reprint requests to Dr. Lim, Division of Derma-tology, University of California Medical Center, San Diego,CA 92103.

Received for publication 19 August 1982 and in revisedform 7 June 1983.

in guinea pigs decomplemented by treatment withcobra venom factor. In contrast to normal guinea pigs,C4-deficient animals exhibited a maximal reaction of0.83±0.16 at 6 h, which subsided within 24 h. Cobravenom factor-treated guinea pigs developed a maximalresponse of 0.5 at 0.5 and at 6 h. These clinical changeswere associated with the development of an increasedvascular permeability, as demonstrated by studies us-ing guinea pigs injected intravenously with Evans bluesolution. In animals with a normal complement system,there was intense localized bluing at the sites of pho-totoxic lesion. In contrast, only minimal bluing wasobserved in decomplemented guinea pigs. These dataindicate that a normal number of polymorphonuclearcells and an intact complement system are requiredfor the full development "of demethylchlortetracy-cline-induced phototoxic lesions.

INTRODUCTION

In previous studies (1, 2), we have demonstrated thatirradiation of normal human serum containing exog-enously added uroporphyrin or protoporphyrin withlight of 400-410 nmwavelengths resulted in activationof the complement system and the generation of che-motactic activity for human polymorphonuclear leu-kocytes (PMN). Similar observations were noted whensera from patients with erythropoietic protoporphyriaor porphyria cutanea tarda were irradiated -in vitro(3). Furthermore, in vivo activation of the complementsystem was noted after irradiation of the forearms ofthese patients (4). These observations suggest that com-plement activation may participate in 'the develop-ment of phototoxic lesions; indirect evidence reported

1326 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/83/10/1326/10 $1.00Volume 71 October 1983 1326-1335

by others also support this concept. Deposition of C3and immunoglobulins around blood vessels and at thedermal-epidermal junction had been observed in light-exposed skin of patients with various types of por-phyria (5). Histologically, exposure of nonlesional skinof patients with erythropoietic protoporphyria to long-wave ultraviolet light resulted in lysis of capillary en-dothelial cells, mast cell degranulation, and the ap-pearance of PMNin the dermis (6). In animals, ad-ministration of an exogenous phototoxic substance,such as chlorpromazine, followed by ultraviolet irra-diation, resulted in dermal edema, vasodilation, andPMNinfiltrate (7). Similar changes were observed bothin guinea pigs that received protoporphyrin intraperi-toneally (2) and in mice rendered protoporphyric bygriseofulvin administration (8). These histologicchanges are consistent with those mediated by prod-ucts of complement activation.

To evaluate whether our previous observation ofphotoactivation of the complement system by uropor-phyrin or protoporphyrin could also occur with otherphototoxic substances, studies were performed usingdemethylchlortetracycline (DMCT),' an exogenousphototoxic agent. In the following experiments, weinvestigated the effect of DMCTand ultraviolet irra-diation on the complement system of human serum.Furthermore, the participation of the complement sys-tem and PMNin the development of phototoxic lesionsinduced by DMCTand ultraviolet light was studied.

METHODSLight source. Four General Electric F40BL tubes (Gen-

eral Electric Co., Medical Systems Div., Milwaukee, WI)were used as the source of ultraviolet-A (UVA) (320-400 nm)light. Photons emitted over a wavelength range of 320-450nm represent 94% of the total energy output of the lamps.With an IL 700 research radiometer (International Light,Inc., Newburyport, MA) and a 3-mm window-glass as a fil-ter, the output of the lamps at 15 cm was as follows: 275-303 nm, 2.13 MW/cm2 (SEE 240 sensor, UVB 63 filter, In-ternational Light, Inc.); 308-318 nm, 13.4 MW/cm2 (PT171C sensor, NB 313 filter); 330-390 nm, 1, 310 MW/cm2(PT171C sensor; WB365 filter); and 400-410 nm, 200 MW/cm2 (PT171 C sensor; NB405 filter). The ultraviolet-B (UVB)(270-320 nm) light source was a barrel of four WestinghouseFS40 tubes (Westinghouse Electric Corp., Bloomfield, NJ),which emit photons over the wavelength range of 275-375nm. The output of the lamps at 15 cm was as follows: 275-303 nm, 307 MW/cm2(SEE 240 sensor, UVB 63 filter); 308-318 nm, 495 MW/cm2 (PT171C sensor, NB 313 filter); 330-390 nm, 680 MiW/cm2 (PT171C sensor, WB365 filter); and400-410 nm, 44 MW/cm2 (PT171C sensor, NB 405 filter).

i Abbreviations used in this paper: CH50, total comple-ment hemolytic activity; DMCT,demethylchlortetracycline;EA, erythrocytes coated with antibody; GVB, veronal-buff-ered saline with 0.1% gelatin; UVA, ultraviolet-A; UVB, ul-traviolet-B.

In vitro irradiation of serum. Pure DMCTpowder (Led-erle Laboratories, Pearl River, NY) was dissolved in phos-phate-buffered saline (PBS), pH 7.4, at a concentration of1.0 mg/ml. 0.1 ml of this stock solution, or serial dilutionsfrom it, was then added to 0.9 ml of normal human serumto yield the final desired concentrations. The ratio of DMCT/serum of 1:9 was used for all the in vitro experiments, unlessit is specifically stated otherwise. To eliminate the effect ofambient ultraviolet irradiation, all procedures involvingDMCTwere carried out under subdued room light. Themixtures were transferred to petri dishes, and placed on ice15 cm away from the light source. Irradiation with UVAlight was then performed, using a 3-mm window-glass toeliminate wavelengths of <320 nm. To evaluate the effectof UVB radiation, sera containing DMCTwere irradiatedin a similar manner with a UVB light source (40 mJ/cm2 at308-318 nm).

Hemolytic titration of total complement activity and in-dividual complement components. Veronal-buffered sa-line, 0.15 M, pH 7.5, containing 0.1% gelatin (GVB); GVBcontaining 0.15 mMCaC12, and 0.5 mMMgCl2 (GVB++);equal volumes of 5% dextrose in water and GVB++(DGVB++); and 40 mMEDTAbuffer in GVB(EDTA-GVB)were prepared as previously described (9). Sheep erythro-cytes were sensitized with rabbit anti-sheep hemolysin(erythrocytes coated with antibody, EA). EACi were pre-pared by mixing EA with human C1, diluted to provide 200effective hemolytic sites/cell. After incubation at 300C for30 min and at 4°C for 15 min, the cells were washed inDGVB++ (10). EAC14b were prepared by incubatingEAC1, 1 X 10' ml, with human C4 to provide 400 effectivemolecules/cell. The mixture was then incubated at 4°C for30 min (11), the cells were then washed and resuspended inDGVB++and stored in the same buffer with penicillin andstreptomycin. EAC4b cells were prepared by incubatingEAC14b cells in 0.04 M EDTA-GVB. These cellular inter-mediates were used to measure total complement hemolyticactivity (CH50) and Cl, C4, C2, C3, and C5 titers (1).

Crossed immunoelectrophoresis. Two-dimensional(crossed) immunoelectrophoresis was performed with minormodifications of the method of Laurell (12). Goat antiserato human C3 were obtained from Atlantic Antibodies, West-brook, ME.

PMN chemotaxis. PMN were obtained from venousblood of healthy adult donors by a previously describedmethod (13). Briefly, PMNwere isolated by dextran-sedi-mentation technique, and suspended in PBS, pH 7.4, sup-plemented with 0.6 mMCaC12, 1.0 mMMgCl2, and 2.0%of bovine serum albumin (wt/vol). Cell suspensions con-tained -85% PMN. Random motility and directed migra-tion (chemotaxis) of these cells were assessed by a modifi-cation of the leading front method of Zigmond and Hirsch,as previously described (1, 13, 14). 2% dilutions of treatedand untreated sera, or buffer, were placed in the lower com-partments of modified Boyden chambers (Nucleopore Corp.,Pleasanton, CA); aliquots (0.8 ml) of leukocyte suspections,containing 2.5 X 106 PMN/ml, were added to the uppercompartments. The upper compartments were separatedfrom the lower ones by 3-um (pore diameter) nitrate cel-lulose micropore filters (Sartorius Filters, Inc., San Francisco,CA). The chambers containing cells and chemoattractantswere incubated at 37°C for 30 min. The filters were thenremoved, fixed in methanol, stained with hematoxylin, de-hydrated in ethanol, and cleared in xylene. The response ofPMN, either to buffer alone (random motility) or to che-motactic stimuli is measured as the distance that the leadingfront of cells migrated into the filters (in micrometers per

Complement and Polymorphonuclear Cells in Phototoxicity 1327

30 min). This was performed by microscopic examinationX 45 objective, measuring the distance from the top of thefilter to the furthest plane of focus that contained at leasttwo cells in focus. For each experiment, duplicate chamberswere used and 10 fields were examined per filter. Goat an-tisera to human C5 used in some of the chemotactic exper-iments were obtained from Meloy Laboratories, Inc., Spring-field, VA.

In vivo induction of localized phototoxic lesions in guineapigs. Albino guinea pigs, 600-800 g, were purchased fromCammResearch Lab Animals (Wayne, NJ). Guinea pigs con-genitally deficient in C4 were obtained from our own colonyat NewYork University Medical Center. The depilated backsof albino guinea pigs were injected intradermally at multiplesites either with 0.1 ml of DMCTin PBS(1.0 mg/ml) or withPBS alone. Only areas below the scapulae were used. 20 minlater, the left side of the backs of different groups of guineapigs were exposed either to UVA (14.4-19.2 J/cm2) or toUVB (40 mJ/cm2), while the right side was covered with alead shield to serve as a nonirradiated control side. Doses ofUVA and UVBchosen were in the range of the known min-imal erythema dose in man for UVA and UVB, respectively.Clinical changes were evaluated serially up to 72 h after thecompletion of the irradiation. These changes were gradedvisually by two independent observers on the following scale:0: no erythema, no induration; 0.25+: minimally perceptibleerythema, no induration; 0.5+: minimal erythema, mini-mally palpable induration; 1.0+: pink erythema, minimalinduration; 1.5+: pink erythema, moderately palpable in-duration; 2.0+: red erythema, moderate induration; 2.5+:red erythema, marked induration; and 3.0+: red erythemawith erosion. For each experiment, the results are expressedas the mean of clinical response score±SEM.

At each time of clinical examination, skin biopsies wereperformed. The tissues obtained were fixed in 10% formalin,embedded in paraffin, cut with a microtome into 6-um-thicksections, and stained with hematoxylin and eosin (15). Thespecimens were examined under light microscope, and theepidermal and dermal changes were noted. The appearanceof neutrophils in the dermis in these specimens were quan-tified by modification of a previously described method (16,17). Briefly, differential cell counts on all infiltrating cells inthe dermis encountered in two randomly selected verticalswaths were performed, using a calibrated optical grid. Asmeasured from the dermal-epidermal junction, each swathwas 156 Mmin width and 474 gm in height. The size of thevertical swath was selected as the dermal changes in thesespecimens were observed within 450 ,m of the dermal-epi-dermal junction. The differential cell counts for each spec-imen were performed on two separate histologic sections;hence, infiltrating cells in a total of four vertical swaths werecounted. The total number of cells counted in the four swathsranged from 300 to 400. Results are expressed as the meanpercentage of neutrophils±SEM. On the basis of the mor-phology and staining characteristics, the following cells weredistinguishable: mononuclear cells, eosinophils, basophils,and neutrophils. Evaluation for the increase in vascular per-meability associated with the development of phototoxic le-sions was performed by assessing the degree of bluing in-duced by 1% Evans blue (Fisher Scientific Co., Fairlawn,NJ) in normal saline. The dye was injected intravenouslyinto the guinea pigs 30 min before the completion of theirradiation, and the degree of bluing was evaluated 30 minlater.

Similar experiments were performed in guinea pigs ren-dered neutropenic or decomplemented, or both neutropenicand decomplemented. Neutropenia was achieved in these

animals by two intraperitoneal injections of cyclophospha-mide (Mead Johnson & Co., Evansville, IN) at 5 and 1 dbefore the study (200 and 50 mg/kg, respectively), a pre-viously described method (18). In pilot studies, we confirmedthat this treatment resulted in a complete depletion of pe-ripheral PMN without any alteration of the complementprofile. Decomplementation was performed by intraperito-neal injection of 300 U/kg of cobra venom factor (CordisLaboratories, Inc., Miami, FL) 16 h before the administra-tion of DMCTand UVA irradiation (18). Our pilot studiesdemonstrated that such treatment resulted in a decrease inthe hemolytic titers of CS and C5 of 90-95 and 55-60%,respectively. Guinea pigs were decomplemented and ren-dered neutropenic by injections of both cobra venom factorand cyclophosphamide.

RESULTS

In vitro effect of DMCT, UVA, or UVB irradiationon human complement system. UVA irradiation (2.4J/cm2) of human serum containing varying concen-trations of DMCT(25-100 #g/ml) resulted in a dose-dependent decrease of CH50, reflecting the decreasein the titers of the components tested: C4, C2, C3, andC5 (Fig. 1). In samples that contained DMCTand werekept in darkness, no alteration of these hemolytic ac-tivities were noted. Exposure of normal human serumto UVA, in the absence of DMCT, did not alter thecomplement profile (Fig. 1, ordinate). Furthermore,the alteration of the complement profile was depen-dent upon the irradiance energy (Fig. 2). To investi-gate whether the decrease in complement hemolyticactivities was due to the nonspecific inhibitory effectof the DMCTmolecules on the complement proteinsor whether it was due to the activation of the com-plement system associated with the generation of bio-logically active cleavage products, serum samples weresubjected to two-dimensional immunoelectrophoresis.The result of a representative experiment is shown inFig. 3. In sample containing DMCT, exposure to UVAresulted in the appearance of an extraanodal precip-itant peak, representing the cleavage products of C3.Such a treatment also resulted in the generation ofpotent chemotactic activity for human PMN(TableI). The chemotactic activity generated was thermosta-ble and was inhibitable by antiserum to C5, but notby antiserum to C3, indicating that it was C5 derived.No chemotactic activity was detected when DMCT-containing sera were kept in the dark. In contrast tothe effect of UVA irradiation, exposure of sera con-taining DMCT(50 ;g/ml) to 40 mJ/cm2 of UVB lightdid not result in alteration of the complement activity(data not shown).

Clinical relevance of the above observations wasstudied in sera containing 5 ;&g/ml of DMCT, a con-centration comparable to that observed in serum ofindividuals taking this medication (19). UVA (16 J/

1328 H. W. Lim, H. Novotny, and I. Gigli

C.,

ui e\...C4-j CHSOH5a 260'*'2Y6

E 4g *- .2C5 _*C3

CD20

25 50 75 100 25 50 75 100

SERUMOMCTCONCENTRATION(agg/ml)

FIGURE 1 Effect of UVA (2.4 J/cm2) and varying serum concentration of DMCTon CH50,C4, C2, C3, and C5 titers. At 25, 50, and 100 Aig/ml DMCT, irradiation resulted in statisticallysignificant decrease of titers of CH50, C2, C3, and C5 (P < 0.05, vs. titers of samples thatreceived PBS and kept unirradiated).

cm2) irradiation of these sera resulted in a decrease ofCH50, Cl, C4, C2, and C3-9 titers (Table II).

The possibility of the generation of a stable photo-product of DMCTand its role in the decrease in he-molytic activity of complement were studied in thefollowing experiment. A solution of DMCT, 50 ug/ml,was exposed to UVA irradiation (24.0 J/cm2) beforeits addition to serum (DMCT/serum, 1:1). As shownin Table III, the CH50 titer was not altered in serumcontaining preirradiated DMCT; furthermore, subse-quent irradiation of this mixture did not result in anysignificant decrease of the titer. In the same experi-ment, serum containing DMCTthat was not preirra-diated had a significant decrease of its hemolytic ac-tivity upon subsequent irradiation. These data indicate

,-NO DMCT100

80

E 60 CH5O_jco 9 ~~~~~C3

° ae 40_1 12 C5

20

2.4 4.8 7.2 9.6UVA, J/Cm2

FIGURE 2 Dose-response effect of UVA irradiation on CH50,CS, and C5 titers of serum samples containing 25 gg/mlDMCT. Irradiation with 2.4, 4.8, 7.2, and 9.6 J/cm2 of UVAresulted in statistically significant decrease of CH50, C3, andC5 titers (P < 0.05, vs. titers of samples that received PBSand kept unirradiated).

(I

(A) NORMALSERUM± IRRADIATION

NORMALSERUM+DMCT

IRRADIATED

NORMALSERUM+DMCT

NON- IRRADIATED

NORMALSERUM(D) + ZYMOSAN

FIGURE 3 Effect of UVA irradiation on electrophoretic mo-bility of CS, as detected by two-dimensional immunoelec-trophoresis technique. Normal serum, with or without ir-radiation, (A), or DMCT-containing serum, kept unirra-diated (B), both demonstrate a single precipitant peak,representing that of native C3. Panel Cshows that irradiationof DMCT-containing serum resulted in the appearance ofan extraanodal precipitant peak, similar to that observedwhen zymosan-activated serum was used (D). This anodalpeak represents cleavage products of C3.


TABLE IGeneration of Chewmotactic Activity in Sera Containing

DMCTand Exposed to UVA

Stimulus PMNmigration'

jsM/30 min

DMCT(100 lsg/ml) + UVA (2.4 J/cm2) 117.2±3.61DMCT(100 jsg/ml) + dark 99.8±2.7DMCT+ UVA - 56°C 115.9±2.31DMCT+ UVA - A-CS 113.1±2.2tDMCT+ UVA - A-C5 98.6±3.1Buffer 96.6±2.7

° Results represent the mean of two experiments±SEM. Duplicatechambers were used in each experiment and 10 fields were ex-amined in each filter.t P, vs. buffer, < 0.05 (Student's t test).

that a stable photoproduct of DMCTwas not involvedin this reaction. In addition, they also suggest thatpreirradiation of DMCTresulted in photodegradationof the majority of DMCTmolecules, rendering themincapable of activating the complement system.

Participation of complement and PMNon the in-duction of phototoxic lesions in an animal model.Intradermnal injection of 0.1 ml DMCT(1.0 mg/ml)on the depilated backs of guinea pigs, followed byexposure to 14.4-19.2 J/cm2 UVA, resulted in the de-velopment of erythema and induration at the sites ofinjection, which peaked at 20 h after irradiation (Fig.4). Histologic alterations consisted of dermal edemaand engorgement of capillaries with erythrocytes at1 h, followed by an increased number of PMNin thedermis, which reached a maximum at 20-24 h andpersisted for at least 72 h. At 24 h, the dermal infiltrateconsisted of 29±2% of neutrophils. Necrosis of the epi-dermis was noted at 20 h, followed by its gradual re-generation. Guinea pigs injected with DMCTand keptunirradiated developed only 0.5 clinical changes at 24

h, which subsided by 72 h (Fig. 4). Histologic changesat these sites were limited to a sparse neutrophilic in-filtrate in the dermis (2.9±0.4% neutrophils at 24 h).At 24 h, the difference between the percentage of neu-trophils at sites injected with DMCTand exposed toUVA and that at sites injected with DMCTand keptin the dark was statistically significant (P < 0.01). Atsites injected with buffer and subsequently irradiatedor at sites exposed to irradiation alone, no clinicalchanges were noted at 24 h. Histologically, there wassome dermal edema at 1 h, which subsided by 24 h.The dermal cellular infiltrate in these specimens con-sisted of <0.5% of neutrophils (range: 0-0.4±0.35%),which was similar to that observed in tissues obtainedfrom untreated sites.

In contrast to the clinical and histologic changesobserved in DMCT-treated guinea pigs exposed toUVA, irradiation with UVB (40 mJ/cm2) induced only0.5±0.25 clinical changes. Histologic findings werelimited to dermal edema at 2 h, followed by the ap-pearance of necrotic keratinocytes at 24 h.

The role of PMNin the development of DMCT-induced phototoxic lesions was studied in guinea pigsrendered neutropenic by cyclophosphamide injections.In these guinea pigs, a maximal clinical response of0.75±0.6 at 24 h was elicited (Fig. 5). This was asso-ciated histologically with the appearance of few intactand fragmented neutrophils in the dermis (1.2±0;5%neutrophils at 24 h). The degree of neutrophilic infil-trate was not statistically significantly different fromthat observed at sites injected with DMCTalone.

The participation of the complement system in thisreaction was studied in guinea pigs congenitally de-ficient of C4, which had normal CS and CS levels.Only the white areas were used in these tricoloredguinea pigs. There was a marked suppression of clin-ical response induced by DMCTand UVA (Fig. 5).Histologically, marked dermal edema, and dilatedcapillaries engorged with erythrocytes were observed

TABLE IIIn Vitro Effect of UVA Irradiation (16j/cm2) on Complement Activities

of Serum Containing 5 eg/ml of DMCT-

Residual complement activityt

DMCTconcentration CH50 C1 C4 C2 C3-9

sg/ln %

5.0 78±2§ 59±15 84±3§ 70±1i 85±110 98±2 98±2 100±1 100±2 100±1

DMCT/normal human serum, 1:9.t Calculated based on titers of samples that received PBS and were kept unirradiated.Values are means±SEM.§ P < 0.05, vs. titers of samples that received PBS and were kept unirradiated.


TABLE IIIIn Vitro Effect of Preirradiated DMCTon CH50

Residual CH50 activitytFinal concentration

Reagent UVA in sera' Dark 2.4 J UVA/cm'

J/cm' pg/ml

DMCT, 50 Ag/ml 24.0 25 99±1 97±2DMCT, 50 Ag/ml 0 25 100±0.5 70±3§

DMCT/normal human sera, 1:1.I Calculated based on titers of the samples that received PBSand were kept unirradiated.Values are means±SEM.§ P < 0.05, vs. titers of samples that received PBS and were kept unirradiated.

at 6 h; at 24 h, only a slight engorgement of capillarieswas noted. The dermal infiltrate consisted of 2.9±0.1%of neutrophils at 24 h, which was similar to that ob-served at sites injected with DMCTalone.

The involvement of the complement system was fur-ther studied in normal guinea pigs depleted of C3 andCSby intraperitoneal injections of cobra venom factor.In contrast to guinea pigs with a normal complementsystem, DMCTand UVA induced a minimal photo-toxic response,which subsided by 24 h (Fig. 5). Theseclinical changes were associated histologically with thepresence of 6.5±0.8% of neutrophils in the dermis at6 h. The neutrophilic infiltrate was not statisticallysignificantly different from that noted at sites injectedwith DMCT.

In guinea pigs injected with both cobra venom factorand cyclophosphamide (hence, C3-C5-depleted as wellas neutropenic), the clinical and histologic changes in-duced by DMCTand UVA were essentially abolished.

Since edema was one of the early findings in thedevelopment of phototoxic lesions, the alteration invascular permeability was assessed in studies using

3+

co

cozCocmC:

2+

1+

rmLffL

guinea pigs injected intravenously with Evans bluesolution. In animals with a normal complement system,intense bluing was noted at sites injected with DMCTand exposed to UVA, reflecting a localized increase invascular permeability (Fig. 6). In contrast, in guineapigs depleted of both C3 and CS, only minimal bluingwas observed. The specificity of both this inhibitionwas demonstrated by the reaction to a topically appliedprimary irritant (croton oil). The response observedin decomplemented guinea pigs was comparable tothat noted in those with a normal complement system.

DISCUSSION

The interactions of phototoxic substances and theirbiologic substrates have been shown to be mediatedby several types of photochemical reactions (20). Thefirst one involves the absorbance of photons by thephototoxic substances, resulting in the formation of theexcited state molecules, which may interact directlywith their biologic substrates to form photoproducts.An example of such a photochemical reaction is the

*

5 min 30 min 6 h 20 h

TIME

1-l*I-I

24 h,

I*]

prL48 h 72 h

FIGURE 4 Phototoxic response induced by DMCT(1.0 mg/ml) and UVA (19.2 J/cm2) in normalguinea pigs. (0), UVA; (O), dark. °P vs. dark < 0.05.

Complement and Polymorphonuclear Cells in Phototoxicity 13t31

0 NEUTROPENIC

a C4-OEFICIENT0 DECOMPLEMENTEDo NORMAL

LUJ

C0cm

c-cc

TIME (hours)

FIGURE 5 Phototoxic response induced by DMCTand UVAin guinea pigs that were neutropenic, or deficient in C4, ordecomplemented. Response observed in normal guinea pigsis shown for comparison. At least three guinea pigs wereused for each experiment. P vs. normal < 0.05.

light-induced formation of a photoadduct of 8-meth-oxypsoralen and a pyrimidine base in the DNAmol-ecule (21). In the second type of reaction, the mole-cules absorb photons to form stable photoproducts,which are toxic to the biological substrates. Both chlor-promazine and protriptyline had been shown to formsuch toxic photoproducts, which caused lysis of eryth-rocytes in vitro (22). A third type of reaction involvesthe generation of toxic oxygen species, such a singletoxygen, superoxide anions, and hydroxyl radicals. Inthis reaction, the molecules that initially absorb thephotons transfer the energy to oxygen molecules, re-sulting in the formation of an excited state of oxygen.Examples of this type of reaction are the toxic oxygenspecies produced by irradiation of protoporphyrin(23), xanthene dyes (24), and thiazine dyes (24).

The elucidation of the above photochemical reac-tions contributes toward the basic understanding of thepathogenesis of cutaneous phototoxic lesions; however,these studies were performed in a serum-free envi-ronment. The role of proteins in the serum, either asthe primary mediator or as the amplifying system, inthe development of phototoxic tissue damage in vivo,has not been investigated. Wehave demonstrated pre-viously that in vitro, as well as in vivo, photoactivationof the complement system occurred in the presenceof uroporphyrin or protoporphyrin, two endogenousphototoxic substances (1-3). This was associated withthe generation of chemotactic activity for humanPMN. These findings suggest that the complement sys-tem participates in the development of cutaneous le-sions in phototoxicity. Such a concept is consistent withthe observations that complement proteins and im-

munoglobulins were deposited in the exposed skin ofpatients with porphyrias (5). In addition, in patientswith erythropoietic protoporphyria, an acute photo-sensitivity flare is associated clinically with burningsensation, edema, erythema, and histologically withthe appearance of PMNin the dermis (6). Further-more, irradiation of forearms of patients with eryth-ropoietic protoporphyria or porphyria cutanea tardaresulted in the activation of the complement sys-tem (4).

To investigate whether other phototoxic substancesare also capable of inducing photoactivation of com-plement, in vitro and in vivo studies were performedwith DMCT, an exogenous phototoxic agent, with anexcitation spectra in the UVA range (25). The dosesof UVA used in these studies were between 2.4 and19.2 J/cm2. Solar irradiance for the wavelength rangeof 330 to 390 nm at sea level on a midsummer day is4,150 ,gW/cm2, which is more than three times greaterthan the output of the light source used (1,310 uW/cm2) (26). Therefore, during the course of daily activ-ities, exposure to equivalent or higher doses of radia-tion energy as those used in these experiments couldbe achieved.

In vitro, irradiation of DMCT-containing sera re-sulted in diminution of CH50 and decrease in C4, C2,C3, and C5 titers (Fig. 1). These changes were depen-dent on the concentration of DMCT, as well as on theirradiance energy administered (Figs. 1 and 2). Asshown in Fig. 3, the diminution of complement he-molytic activity was associated with the alteration inthe electrophoretic mobility of C3, presumably owingto the appearance of cleavage products. Furthermore,C5-derived chemotactic activity for human PMNwasgenerated (Table I). In contrast to UVA, UVB irra-diation of such sera did not result in complement ac-tivation. This is consistent with the known excitationfluorescence spectra of DMCT, as well as its clini-cal action spectra, which are both in the range ofUVA (25).

The clinical relevance of the above observations wasdemonstrated in experiments using sera containingDMCTat a concentration achieved clinically (5 ,ug/ml) (19). As shown in Table II, irradiation of serumcontaining 5 gg/ml of DMCTalso resulted in pho-toactivation of the complement system.

The possible role of stable photoproduct(s) of DMCTthat might induce the activation of the complementsystem was investigated in experiments summarizedin Table III. Exposure of DMCTto UVA light beforeits addition to normal human serum did not cause anyalterations of the complement profile, indicating thata stable photoproduct was not involved in this reaction.Furthermore, such preirradiation of DMCTresultedin the loss of its efficacy to activate the complement


W.~~~~~~~~~~~~~~~~~..

FIGURE 6 Clinical appearance of a representative guinea pig 30 min after intravenous injectionof Evans blue solution. The back of this animal was injected with DMCTat multiple sites; theleft side of the back was then exposed to UVA, while the right side was protected from theirradiation. Intense bluing is observed only at sites injected with DMCTand exposed to UVA.

system upon subsequent irradiation, presumably ow-ing to photodegradation of the molecules.

Having demonstrated that in vitro irradiation ofDMCT-containing serum with UVA resulted in theactivation of the complement system and in the gen-eration of chemotactic activity for PMN, we investi-gated the role of complement and neutrophils in thedevelopment of phototoxic lesions in vivo, using guineapigs as an animal model. Intradermal injection ofDMCT, 100 jLg/site, followed by UVA irradiation, con-sistently resulted in the development of phototoxic le-sions, as manifested by cutaneous erythema and edema,whereas only minimal erythema and edema was notedat the DMCTinjected, nonirradiated sites (Fig. 4). The

quantity of DMCTinjected was chosen on the basisof a study of tetracycline-induced phototoxicity inwhich the concentration of the drug in the involvedskin was found to be 10-fold higher than that in theserum (27). Clinical evaluation of the evolution of thephototoxic response showed that it reached its peak 20h after the completion of the irradiation and then sub-sided gradually. Histologically, there was dermaledema at 1 h, followed by PMN infiltrate, whichpeaked at 20-24 h. At 24 h, the dermal infiltrate con-sisted of 29±2% of neutrophils. These clinical and his-tologic changes are similar to those of phototoxic le-sions induced by intraperitoneal administration ofDMCT(7) or protoporphyrin (2, 28). These results also


confirm and extend the previously reported observa-tion that intradermal injection of DMCTinto backsof mice, followed by "black light" irradiation, resultedin the development of cutaneous erythema (25).

The presence of PMNat the sites of phototoxic le-sions suggested that these cells may contribute to thedevelopment of tissue damage. This was investigatedin guinea pigs rendered neutropenic by intraperitonealinjections of cyclophosphamide (18). Such injectionsresulted in complete depletion of circulating PMNwithout altering the complement profile. In theseguinea pigs, the maximal intensity of the reaction wasdiminished; whereas a response of 2.25±0.25 was ob-served in normal guinea pigs, only 0.75±0.6 responsewas noted in cyclophosphamide-treated animals at 24h (Fig. 5). Histologically, few intact and fragmentedneutrophils (2.9±0.4% at 24 h) were observed in thedermis. As compared with response observed in normalguinea pigs, the suppression of clinical response inneutropenic guinea pigs was statistically significant at6 h and at 48 h; however, it was not statistically sig-nificant at 24 h. These data indicate that although anormal number of neutrophils is apparently requiredfor the full development of phototoxic lesions, a mech-anism independent of neutrophils is also operative.

Since dermal edema is the feature of the early his-tologic lesions, the participation of the complementsystem in the development of phototoxic lesions wasstudied in experiments performed in guinea pigs con-genitally deficient in C4 and in decomplemented an-imals. Clinically, a maximal response of only 0.83±0.16was observed in C4-deficient guinea pigs (Fig. 5). His-tologically, there was marked dermal edema, accom-panied by the appearance of PMN(2.9±0.1% neutro-phils at 24 h). In guinea pigs depleted of C3 and par-tially of C5, a minimal response (0.5) was observed(Fig. 5). These observations were corroborated by stud-ies using intravenously injected Evans blue solution.In guinea pigs with normal complement system, in-tense localized bluing occurred at sites injected withDMCTand exposed to UVA (Fig. 6), whereas the sametreatment induced only minimal bluing in cobravenom factor-treated animals.

There are other mediators of inflammation that havebeen shown to be responsible, at least in part, for thedevelopment of UV-induced erythema. Degranulationof mast cells has been observed after UVB (290-320nm) irradiation of normal skin (29), as well as in tissueobtained from patient with erythropoietic protopor-phyria after exposure to long-wave ultraviolet light(6). Cutaneous erythema induced by UVB, but not thatinduced by UVA (320-400 nm), was inhibitable byindomethacin, an inhibitor of cyclooxygenase pathwayof arachidonic acid metabolism (30, 31). In vitro, fi-

broblasts have been reported to geherate chemoat-tractants for PMNand monocytes (32); however, thein vivo significance of this observation in UV-inducederythema is not clear. Although results of the studieswith cobra venom factor-treated, decomplementedanimals suggest that the complement system plays asignificant role in the development of DMCT-inducedphototoxic lesions, the participation of other mediatorsremains to be elucidated.

The mechanism of complement activation inducedby DMCTand UVA is not clear. A stable photoproductof DMCTprobably was not involved in this process(Table III). Another mechanism that may producephotoactivation of the complement system by DMCTinvolves the absorption of photons by the molecules,converting them into an excited state. The latter mayinteract directly with complement proteins, or withother serum proteins (e.g., C-reactive protein, plas-minogen, Hageman factor, etc.), which in turn acti-vates the complement system. Alternatively, photoac-tivation of complement may also involve the genera-tion of toxic oxygen species. Experiments are inprogress attempting to elucidate this interesting phe-nomenon.

The complement system is one of the principal ef-fector systems of inflammation (33). It had been shownpreviously that porphyrins, upon exposure to 400-410nm light, activated this system in vitro, as well as invivo (1-4). In this report, we demonstrated that thecomplement system is also activated by DMCTin thepresence of UVA irradiation. Therefore, these datasuggest that the previously described photochemicalreactions, which produced cellular damage in a serum-free environment, may also result in the activation ofthe complement system in the presence of serum.Whether the complement system serves as the primarymediator of phototoxic tissue injury in vivo or as anamplifying mechanism remains to be elucidated.

ACKNOWLEDGMENTSThe authors wish to acknowledge Mr. Bruce Weintraubfor technical assistance; Dr. Anna Ragaz for reviewing thehistologic sections; Dr. Maureen Poh-Fitzpatrick, and Dr.Donald V. Beltito for their review of the manuscript; andMs. Elizabeth Drahman for secretarial assistance.

This work was supported by grants from the National In-stitutes of Health (AI 17365, AI 13809, and AM01165) andby the Irma T. Hirschl Trust.

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role of complement and polymorphonuclear cells in

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