Circulating Immune Complexes in Cutaneous Vasculitis

DETECTIONWITH Clq ANDMONOCLONALRHEUMATOIDFACTOR

SUSANE. MACKEL, GERHARDTAPPEINER, HILTON BRUMFIELD, andROBERTE. JORDON,Cutaneous Immunopathology Unit, Research Service,Veterans Administration Center, Wood (Milwaukee), Wisconsin 53193;Dermatology Section, Department of Medicine, Medical Collegeof Wisconsin, Milwaukee, Wisconsin; Department of Dermatology,University of Innsbruck, Austria

A B S T RA C T To investigate the pathogeneic signif-icance of immune complexes in cutaneous vasculitis,107 patients with various forms of cutaneous vasculitis,including 59 patients with necrotizing (leukocytoclas-tic) vasculitis (group 1), and 48 patients with lympho-cytic vasculitis, or a predominately lymphocytic peri-vascular infiltrate (group 2), were studied. Immuno-globulins or complement components in cutaneousblood vessels were detected by direct immunofluores-cence in high frequency in both groups (91 and 88%,respectively). Using two radioassays for circulatingimmune complexes, Clq or monoclonal rheumatoidfactor (mRF) reactive material was detected in 68%of the patients with necrotizing vasculitis but only 44%of the patients in the lymphocytic-perivascular group.The mRFradioassay was elevated in 58% of the firstgroup of patients and 41% of the patients in group 2,although Clq binding activity was increased in 54%of the patients with necrotizing vasculitis but only in9% of the patients with a lymphocytic vasculitis orlymphocytic perivascular infiltrate. By using bothsucrose density gradient ultracentrifugation and Sepha-rose 6B gel filtration, the Clq and mRFreactive ma-terial detected in some patients with necrotizing vas-culitis eluted in high molecular weight fractions thatwere also anticomplementary. In one patient withnecrotizing vasculitis and hepatitis B antigenemia, theseheavy molecular weight Clq and mRFreactive frac-tions contained a two- to three-fold increase in hepatitis

Presented in part at the Annual Meeting of the AmericanFederation for Clinical Research, 30 April 1978, San Fran-cisco, Calif.

Dr. Mackel is a recipient of U. S. Public Health Service Fel-lowship 1F32 AM0637-01. Dr. Jordon is a recipient of aMedical Career Investigatorship of the Veterans Administra-tion.

Received for publication 26 March 1979 and in revisedform 19 July 1979.

1652

B surface antigen when compared with lighter molecu-lar weight fractions. Heavy and light molecular weightmRFreactive material could be detected in selectedpatients in the lymphocytic-perivascular group as wellas in the necrotizing vasculitis group. These studiessuggest that cutaneous vasculitis, including acute nec-rotizing (leukocytoclastic) vasculitis and some formsof lymphocytic vasculitis, and perhaps some diseasescharacterized by a lymphocytic perivascular infiltrate,may represent cutaneous expressions of immune com-plex disease.

INTRODUCTION

Cutaneous necrotizing vasculitis is an inflammatorydisease of dermal blood vessels that presents clinicallyas palpable purpura located primarily on the lowerextremities (1, 2). Histologically, the hallmark of thisdisease is an infiltration of blood vessels by polymor-phonuclear leukocytes resulting in a fibrinoid necrosisof the vessel walls with endothelial cell swelling, ex-travasation of erythrocytes, and leukocytoclasis (3-5).Although immunopathogenic mechanisms have beenpostulated for this disease, the evidence to substantiatethis theory is still inconclusive.

The histologic changes seen in necrotizing vasculitisresemble those found in necrotic lesions produced bythe Arthus phenomenon (6), a localized antigen-anti-body reaction (7). Direct immunofluorescence (IF)' ofskin biopsy specimens often shows the deposition ofimmunoglobulins, complement components, and fibrinin the walls of affected blood vessels (6, 8-11). Theseobservations and the demonstration of anticomple-

'Abbreviations used in this paper: Agg, heat-aggregated;BA, binding activity; CH,o, hemolytic complement; HBsAg,hepatitis B surface antigen; IF, immunofluorescence; mRF,monoclonal rheumatoid factor; RF, rheumatoid factor.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/79/12/1652/09 $1.00Volume 64 December 1979 1652-1660

mentary activity (12-14) in the sera of some patientshave led to the hypothesis that necrotizing vasculitisrepresents a cutaneous expression of immune complexdisease. This theory is supported clinically by the in-creased frequency of both hypocomplementemia (15-18) and cryoglobulinemia (12-14, 19) in these patientsas well as an increased occurrence of various forms ofcutaneous vasculitis in patients with known immunecomplex-associated diseases, for example, systemiclupus erythematosus. Further, Braverman et al. (20)have demonstrated the existence of electron dense ma-terial in the blood vessels of histamine-induced lesionsin patients with active necrotizing vasculitis beforeinfiltration of polymorphonuclear cells; deposits thatcorresponded to deposition of immunoglobulin and C3detected by direct IF.

Despite the fact that circulating immune complexeswith subsequent deposition in cutaneous vessels hasbeen postulated as the pathogenetic mechanism innecrotizing vasculitis, few reports have appeared inthe literature to document this basic hypothesis. Inan attempt to add support to the theory of an immunecomplex-related pathogenesis, serum samples from 107patients with various forms of cutaneous vasculitis, in-cluding 59 patients with cutaneous necrotizing (leuko-cytoclastic) vasculitis were tested for the presence ofcirculating immune complexes using both Clq bindingand monoclonal rheumatoid factor (mRF) inhibitionradioassays. Patients with a lymphocytic vasculitis andpatients who demonstrated primarily a lymphocyticperivascular infiltrate were included in the study as agroup for comparison with patients with necrotizingvasculitis. To characterize the reactive material presentin the sera as immune complexes, density gradientultracentrifugation and gel filtration chromatographytechniques were performed on selected samples. Frac-tions were then tested for the presence of immunecomplexes by both radioassays, a standard anticom-plementary assay, and by double immunodiffusion.

METHODSReagents. Reagents used in this study included: Sepha-

rose 6B, Sephadex G-25, and Sephadex G-200 (Pharmacia FineChemicals, Uppsala, Sweden) antibodies to immunoglobulin(Ig)G, IgM, IgA, C3 and fibrin (Meloy Laboratories, Inc.,Springfield, Va.); IgG Cohn fraction II (Research ProductsDivision, Miles Laboratories, Inc., Elkhart, Ind.); disodiumethylenediamine-tetraacetate (EDTA), ACS certified sucrose(Fisher Scientific Co., Pittsburgh, Pa.); CnBr (Eastman KodakCo., Rochester, N. Y.); polyethylene glycol 6000, agarose, lac-toperoxidase (Sigma Chemical Co., St. Louis, Mo.); avicelmicrocystalline cellulose (Brinkmann Instruments, Inc., West-bury, N. Y.); DNAand DNase I (Worthington BiochemicalCorp., Freehold, N. J.) and 125I (New England Nuclear,Boston, Mass.).

Patient selection. The patients in this study were selectedon the basis of clinical and histologic findings. The lesionsof vasculitis were polymorphic. Necrotizing vasculitis typi-cally presented as palpable nonblanching erythematous

papules with a tendency to ulcerate. The lesions were lo-cated primarily on the lower legs and feet. On routine his-tologic examination, the walls of the small dermal blood ves-sels were infiltrated by polymorphonuclear leukocytes. Neu-trophils or a mixed pattem of neutrophils and mononuclearcells surrounded the involved vessels. There was evidenceof leukocytoclasis (fragmentation of nuclei) in the surroundingtissue. Fibrinoid necrosis of the vessel wall was also apparent.Extravasation of erythrocytes and endothelial cell swellingwere often present and hemosiderin deposits were occasion-ally noted. Subcutaneous nodules and ulcerations were seenin patients with cutaneous periarteritis nodosa and histologicexamination of these lesions revealed vasculitis and necrosisof larger, deeper dermal blood vessels.

Most patients with a lymphocytic vasculitis or lymphocyticperivascular infiltrates had clinical lesions typical of erythemamultiforme, livedoid vasculitis (atrophie blanche), or acuteparapsoriasis. In erythema multiforme the most characteristichistologic finding was a mononuclear perivascular infiltrate.In patients with atrophie blanche there was endothelial cellproliferation with thickening and thrombosis of small dermalvessels. These vessels were surrounded by an infiltrate com-posed of lymphoid cells and histiocytes. Acute parapsoriasis(Mucha-Habermann) was characterized by a perivascular in-filtrate of mononuclear cells with endothelial cell swellingand focal erythrocyte extravasation. The walls of these vesselswere often permeated by the infiltrating cells, but neutrophilsand leukocytoclasis were not present.

In most cases the etiology of the cutaneous vasculitis couldnot be determined. Some lesions were thought to representhypersensitivity reactions to drugs, whereas others were as-sociated with an underlying systemic disorder. Diseases af-fecting some of the patients with necrotizing vasculitis in-cluded rheumatoid arthritis, angioimmunoblastic lymphade-nopathy, subacute bacterial endocarditis, Hodgkin's disease,hypereosinophilic syndrome, and cryoglobulinemia.

Sera for immune complex studies were separated fromfreshly drawn blood and kept frozen at -70°C until use. Se-quential serum samples were obtained whenever possible.One full thickness punch biopsy of lesional skin was takenfrom each patient for histologic study after local anesthesiawith 2% lidocaine. The tissue was fixed in 10% formalin solu-tion, stained with hematoxylin and eosin, and examined bylight microscopy. Early lesions (<24 h old) were biopsiedwhenever possible.

Direct immunofluorescence. Skin biopsy specimens fromearly lesions were obtained for direct IF at the time of biopsyfor histologic study. Specimens were frozen immediately inliquid nitrogen and stored at -70°C until sectioned. DirectIF staining was performed by methods previously describedin detail (21) using monospecific fluorescein-conjugated anti-sera to IgG, IgM, IgA, C3, and fibrin. Antisera were checkedfor activity and specificity by immunodiffusion (Ouchterlony)and immunoelectrophoresis. Units of antiserum, antibodyprotein assays, fluorescein-protein ratios, and dilutions atwhich these conjugated antisera were used, conformed to pre-viously determined standards (21).

Clq binding assay. The Clq binding assay.was performedby the method of Nydegger et al. (22) with modifications byTappeiner et al. (23) and Zubler et al. (24). Briefly, Clq wasisolated from defibrinated fresh human plasma by the DNAextraction method of Agnello et al. (25), and labeled with125I in a lactoperoxidase-catalyzed reaction (26).

IgG (25 mg/ml) was heated at 63°C for 12 min and separatedby gel filtration on Sepharose 6B in 0.1 M Tris-HCl, 0.2 MNaCl, pH 8.5 (23). Only protein eluting in the exclusion vol-ume was used as heat-aggregated (Agg) IgG.

To assay Clq binding activity (BA), 0.2-ml aliquots of serum

Immune Complexes in Cutaneous Vasculitis 1653

sample were diluted in 0.2 ml of 0.26 MEDTA, pH 7.5. Toeach tube, 0.05 ml of a 5-,ug/ml solution of 1251-C lq was added.After incubation, 3 ml of a solution of polyethylene glycol6000 was added to make a final concentration of 1.5% poly-ethylene glycol. After 2 h incubation at 40C, the tubes werecentrifuged, supernates discarded, and the remaining reactiv-ity measured in a gammascintillation spectrometer (PackardInstrument Co., Inc., Downers Grove, Ill.) and calculated aspercent of the total activity added. Results were comparedwith binding of known concentrations (5-1,000 ,Ag) of AggIgG and were expressed as micrograms of Agg IgG equivalentsper milliliter. Normal levels of Clq BA were <5 ,ug AggIgG eq/ml.

mRFinhibition assay. Two radioassays that use mRFwereperformed using methods described by Luthra et al. (27) andGabriel and Agnello (28). These different assays were per-formed to test for the possibility that in the solid-phase mRFassay (27), rheumatoid factor present in the patients' sera couldbind to 1251-labeled Agg IgG resulting in lower counts andfalse positive test results.

In the solid-phase mRFassay, mRFwas separated fromthe serum of a patient with macroglobulinemia. This serumwas dialyzed against 0.1 M glycine HC1, pH 3.0 buffer for12 h at 40C, and applied to a Sephadex G-200 column in thatbuffer. The exclusion protein peak was conjugated with CnBrto microcrystalline cellulose beads.

IgG Cohn fraction II was aggregated by heating at 630Cfor 12 min and was then applied to a sucrose density gradientto obtain aggregates >25S. These aggregates were radiola-beled with 125I, again in a lactoperoxidase-catalyzed reac-tion (26).

Tubes containing 0.5 ml serum sample dilution, 0.5 ml 125I-Agg IgG dilution (0.4 ,ug/ml), and 0.5 ml mRF-cellulose dilu-tion were incubated with rotation, centrifuged, washed withbuffer, then counted in a gammascintillation spectrometer.Results were compared with a standard curve of Agg IgG(0.03-4 ,ug/ml) in normal human serum and expressed as micro-gram Agg IgG equivalents per milliliter. Levels >5 jig AggIgG eq/ml were considered abnormal.

In the soluble mRFassay (28), mRFwas separated as de-scribed above and labeled with 25I by the method of Heusseret al. (26). IgG was coupled to Sepharose 6B by the methodof Bing (29) using CnBr. After incubation of 0.1 ml 125I-mRFwith 0.01 ml serum sample and buffer (0.075 M Tris-HCl,0.01 MNa2H2 EDTA, 1%bovine serum albumin, pH 7.4) 0.1ml IgG-Sepharose was added. The tubes were then incubated,centrifuged, and washed. The radioactivity of the remainingpellet was counted in a gammascintillation spectrometer andthe results were again expressed as micrograms of Agg IgGequivalents per milliliter after comparison with a standardcurve of Agg IgG prepared as described above.

Density gradient ultracentrifugation. Undiluted serumsamples of 0.5 ml were layered over a 5-40% sucrose densitygradient in Beckman polyallomer tubes (Beckman Instru-ments, Inc., Fullerton, Calif.) and centrifuged at 217,500 gfor 15 h at 4°C in a Beckman L5-50 preparative ultracentrifugewith a SW-40TI rotor. The gradient tubes were puncturedafter centrifugation, and 14 fractions, -0.65 ml each, werecollected.

Gelfiltration chromatography. Undiluted serum samplesof 1.0 ml each were passed over a column of Sepharose 6B(2.0 x 60 cm, downward flow, 0.1 MTris-HCl, 0.2 M NaCl,pH 8.5 buffer at 4°C). Fractions were collected every 2 minwith a flow rate of 30 ml/h. Effluents were monitored for pro-tein content by measuring the optical density at 280 nm.

Anticomplementary activity. Anticomplementary activitywas assayed by methods previously described (30). Briefly,0.025-ml aliquots of serum that had been fractionated by either

density gradient ultracentrifugation or gel filtration (see above)were diluted (1:2-1:256) in a microtiter plate in 0.025 mldextrose gelatin veronal buffer. Using a dilution just capableof producing 100% lysis, normal human serum (0.025 ml) wasadded to each well as a source of complement. After incuba-tion of plates at 30°C for 30 min, 0.025 ml sensitized sheepcells (EA 1.5 x 108/ml) was added. The plates were then in-cubated for 1 h at 37°C, centrifuged, and read for hemolysis.

Miscellaneous. Protein concentration of density gradientfractions was estimated by the Hartree modification (31) ofthe Lowry method (32). Cryoproteins were detected by meth-ods previously described (33). Total serum hemolytic com-plement (CHO) was determined by a modification of themethod of Shulman (34). Double immunodiffusion (Ouchter-lony) was carried out in 0.6% agarose in veronal-bufferedsaline, pH 7.5. Plates were developed at 370C and read after24 and 48 h. Hepatitis B surface antigen (HBsAg) was assayedby solid-phase radioimmunoassay (Abbott Diagnostics, NorthChicago, Ill.).

RESULTS

84 of the 94 biopsy specimens showed positive directIF of blood vessels with anti-immunoglobulins, C3,or fibrin. Fig. la demonstrates blood vessel IF afterstaining for fibrin, a pattern that is characteristic forleukocytoclastic vasculitis. Fig. lb demonstrates directIF of blood vessel walls with antiserum to IgM. Speci-mens from 45 of 54 patients with leukocytoclastic vas-culitis showed vessel immunoglobulin or complementdeposits, and 26 of 40 patients with lymphocytic vas-culitis or a lymphocytic perivascular infiltrate demon-strated similar findings.

A combination of deposits of IgM, C3, and fibrinwas detected in 24 of the 94 specimens. IgA was foundin 15 biopsies and IgG in only 3, all from patientsin the leukocytoclastic vasculitis group.

Specimens from three patients with Henoch-Schoen-lein purpura demonstrated IgA deposition in bloodvessels, a finding previously described in such patients(35). In this disease various combinations of cutaneousvasculitis with signs of systemic vasculitis, includingarthritis, abdominal pain, melena, and hematuria arefrequently seen. Two of our patients were childrenwho developed a purpuric eruption, fever, arthralgias,and abdominal pain or nausea after a sore throat. Onechild later developed gl6merulonephritis. The thirdpatient, an adult, had four episodes of a purpuric rashassociated with arthritis, abdominal pain, and melena.

Cryoglobulins and depression of total complement.70 serum samples were tested for the presence of cryo-globulins. Cryoglobulins were detected in 15 of 52 pa-tients with leukocytoclastic vasculitis but in only 2 of18 patients with lymphocytic vasculitis or a lympho-cytic perivascular infiltrate.

14 of 72 patients tested had depressed CH. levels.All of these patients had leukocytoclastic vasculitis.Of the 17 patients with measurable cryoglobulins, 6also had low CH,,.

1654 S. E. Mackel, G. Tappeiner, H. Brumfield, and R. E. Jordon

FiGuRE 1 (a) Direct IF with fluorescein-conjugated anti-fibrin demonstrating diffuse IF in and around cutaneous bloodvessel. x400. (b) Superficial cutaneous blood vessels demon-strating IgM in vessel walls (arrows). x250.

Clq bindinig assay. Serum samples from 107 pa-tients with cutaneous vasculitis were tested for thepresence of circulating immune complexes by the Clqbinding assay. A total of 36 patients (34%) had serareactive with Clq. 32 of 59 patients (54%) in the leuko-cytoclastic vasculitis group but only 4 of 48 (9%) inthe lymphocytic-perivascular group demonstrated ele-vated Clq BA (Fig. 2). 19 patients with leukocytoclasticvasculitis demonstrated markedly elevated levels of

Clq BA, whereas only 1 patient in the lymphocytic-perivascular group showed Clq BA significantly ele-vated from normal. Of the 26 patients with -erythemamultiforme, samples from only 2 were reactive withClq. These patients had levels of Clq BA of 6 and 12,ug Agg IgG eq/ml. None of the 150 control sampleshad elevated Clq BA.

mnRF assays. The solid-phase mRFassay (27) waspositive in 49 of 101 (49%) serum samples tested. 30 of

Immune Complexes in Cutaneous Vasculitis 1655

4c

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CONTROLS LEUKOCYTOCLASTIC LYMPHOCYTIC]

50

0/150 32/59 4/48POSrIVE POSITIVE POSITIVE

40 -

30-

20

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:'......... ...._.O- ;.............................. ......................... ..............-

FIGuRE 2 Scattergram demonstrating Clq reactivity in serumsamples from patients with leukocytoclastic vasculitis andlymphocytic vasculitis or a perivascular lymphocytic infiltrateand in normal control sera. Serum samples with normal levelsof Clq BA (c5 ,ug Agg IgG eq/ml) are included in the hatchedareas.

55 samples (55%) from patients with leukocytoclasticvasculitis had elevated levels (Fig. 3). Interestingly,19 of 46 serum samples (41%) from patients in thelymphocytic-perivascular group also were positive. 35control samples were negative.

38 samples from 23 patients with cutaneous vasculitiswere tested by the soluble mRFassay (28). 9 patientshad a lymphocytic vasculitis or lymphocytic perivas-cular infiltrate and 14 had leukocytoclastic vasculitis.These results were compared with those obtained withthe solid-phase mRFassay (27). There was agreementin 35 of the samples tested. Three samples were posi-tive by the solid-phase mRFassay but negative by thesoluble mRFassay, and only one of these cases showeda significant difference in the level of mRFBA. Inone case, increased binding was detected by the solu-

E

Sv

4c

S.

CONTROLS LEUKOCYTOCLASTIC LYMPHOCYTIC.8.~~~~~~~l

0/35 30/55 19/"50- POSITIVE POSIVE POSIWE

40-

30-

20-

10-

.................___ ..

:-:-:::.:-::--::S::::X: ~ ~ ~ ~ ~::: ::::::-....:::..::::-::2

FIGuRE 3 Scattergram demonstrating mRF reactivity inserum samples from patients with leukocytoclastic vasculitisand lymphocytic vasculitis or a perivascular lymphocyticinfiltrate and in normal control sera. Serum samples withnormal levels of mRFreactive material (c5 ,tg Agg IgG eq/ml)are included in the hatched areas.

ble mRF; assay alone, but only at a minimally elevatedlevel. 11 of the 13 patients tested for the presence ofrheumatoid factor (RF) were negative and 2 were posi-tive. In one of these two patients there was disagree-ment in the results obtained with the two mRFradio-assays. BA was detected with the solid-phase mRFassay, whereas the soluble mRFassay was within nor-mal limits. Because only a small number of RF-positivepatients were tested by both mRFassays, it could notbe determined whether RF that is present in a patient'sserum could bind to the Agg IgG in the solid-phasemRFassay and give a false positive result. However,a comparative study done previously in this laboratory(unpublished data) demonstrated good correlation be-tween these two assays when used in testing patientswith rheumatoid arthritis.

Serial blood samples obtained from several patientswith necrotizing vasculitis showed a rough correlationbetween the level of Clq BA or mRFreactive materialand severity of disease. In one patient, a subsequentonset of hematuria was associated with a marked risein mRFreactive material and a slight elevation of ClqBA. In another patient who had two episodes of palpablepurpura within 2 moa delayed rise in Clq BAand mRFreactive material was seen during each episode. Usuallya lag of 5-7 d between onset of clinical disease anddetection of circulating immune complexes was noted.Peak levels of Clq BAand mRFreactive material weremost often detected when the skin lesions were clear-ing and no new lesions were apparent.

Characterization studies. Normal sera and serafrom eight patients with leukocytoclastic vasculitis, onepatient with a lymphocytic vasculitis, and three patientswith erythema multiforme were selected for study bydensity gradient ultracentrifugation and gel filtrationchromatography.

Fig. 4 summarizes the results of studies performedon fractions obtained by density gradient ultracentrif-ugation in a representative patient with leukocytoclas-tic vasculitis (F.R.). The protein content of each fractioncollected is shown as a solid line with IgM and IgGmarkers as indicated. Immune complexlike materialwas detected in fractions 1, 2, and 3 by both the Clqbinding assay (hatched area) and the solid-phase mRFassay (broken line). Anticomplementary activity waspresent at a dilution of 1:64 in fraction 1 and at 1:128in fractions 2-5.

Solid-phase radioimmunoassay for HBsAg was per-formed on density gradient fractions of patient F.R.who had HB.Ag-positive hepatitis in addition to vas-culitis. HB,Ag was found in high levels in the heavygradient fractions (Fig. 4) when compared with latergradient fractions.

Fig. 5 summarizes the results of studies performedon fractions collected after gel filtration of serum fromE.W., a patient with rheumatoid arthritis, leukocyto-

1656 S. E. Mackel, G. Tappeiner, H. Brumfield, and R. E. Jordon

0in

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FRACTION O 1 2 3 4 5 6 7 8 9 10 11 12

H" 15 12 12 17 21 6 4 4 3 4 4(cpm X 10a)

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FIGURE 4 Sucrose density gradient ultracentrifugation ofserum from patient F.R. Protein content is shown (0) withIgM (19S) and IgG (7S) markers. Anticomplementary activity(*, 4W) was detected in fractions 1-5. The hatched arearepresents fractions containing Clq reactivity. mRFreactivefractions are indicated by a broken line. HBSAg content asmeasured by radioimmunoassay is shown.

clastic vasculitis, and cryoglobulinemia. The opticaldensity (280 nm) of each fraction is shown as well asthe location of IgM and IgG markers. Reactivity withClq was located in fractions 52-56 (hatched area),and the solid-phase mRFassay was positive in frac-tions 52-55 (broken line). Anticomplementary activitywas present in fractions 56-57 at dilutions of 1:32 and1:64, respectively.

Anticomplementary activity was detected in theheavy molecular weight fractions obtained from fiveother patients with necrotizing vasculitis, and reactivitywith Clq and mRFwas demonstrated in these samefractions in four patients. The serum of one patientwith necrotizing vasculitis had high levels of mRFre-active material but no Clq BA. By sucrose density gra-dient ultracentrifugation, mRFreactive material in thisserum eluted in somewhat lighter fractions (fractions10, 11, and 12) -8-1OS in size. Only mRFreactivematerial, however, was detected in heavy molecularweight fractions (19S or greater) in the patients withlymphocytic vasculitis and in one of the three patientswith erythema multiforme.

Normal sera, in addition to pathologic sera, con-sistently showed anticomplementary activity at a 1:16dilution in sucrose density gradient fractions 6 and 7,a phenomenon not well understood. Early fractionsof control sera, however, were always negative for anti-complementary activity. All fractions of normal serawere negative for both Clq and mRFreactive material.No Clq or mRFbinding reactivity or anticomplemen-tary activity was present in fractions obtained from gelfiltration of normal sera.

By using double immunodiffusion, precipitin bandreactions against mRFand Clq could be demonstrated

against gel filtration fractions containing Clq and mRFreactive material with anticomplementary activity (Fig.6a and b). In fractions obtained by density gradientultracentrifugation, only precipitin bands against mRFcould be demonstrated. IgM and, occasionally, IgGwere detected by double immunodiffusion in heavymolecular weight anticomplementary fractions.

DISCUSSION

Leukocytoclastic vasculitis has long been considereda cutaneous form of immune complex disease (13, 16,20). The studies reported herein support this conten-tion as circulating immune complexes were detectedin 32 of 59 serum samples (54%) using the Clq bindingassay and in 30 of 55 (55%) tested by the mRFsolid-phase radioassay. 22 of the serum samples had immunecomplexes detectable by both assays. Thus, in leuko-cytoclastic vasculitis, not only is there considerableindirect evidence, such as a histologic similarity of thecutaneous lesions to the Arthus reaction and IF evi-dence of immunoglobulin and complement depositionin affected blood vessels, but also direct evidence bythe demonstration of anticomplementary material inthe circulation of these patients that is reactive withboth Clq and mRF. In addition, both density gradientand gel filtration studies of most sera suggest that theClq and mRFbinding material in these patients is>19S, a size consistent with antigen-antibody com-plexes (36).

There was no significant difference in the incidenceof positive direct IF in the various subgroups of pa-tients (rheumatoid arthritis, cryoglobulinemia, drugreactions, and systemic disease) with necrotizing vas-culitis. However, Clq reactive material was detectedin a greater frequency than mRFreactive material in

00

li

. 20

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4 a

50 53 66 59 62 65 6O 71 74 7nFRACTION

FIGURE 5 Sepharose 6B gel chromatography of serum frompatient E.W. Protein content is shown (0) with IgM (19S)and IgG (7S) markers. Anticomplementary activity ( i ,-f* *, i)was detected in fractions 55-59. The hatched area representsfractions containing Clq reactivity. mRF reactive fractionsare indicated by a broken line.

Immune Complexes in Cutaneous Vasculitis 1657

58mRF

AGG

57FIGURE 6 (a) Gel diffusion showing precipitin bands against Clq in Sepharose 6B fractions(E.W.) 52,54, and 58. (b) Gel diffusion demonstrating precipitin bands with mRFand Sepharose6B fractions (E.W.) 56 and 57.

the sera of patients with both rheumatoid arthritis andcryoglobulinemia. In contrast, mRFreactive materialwas detected more often than Clq reactive materialin the idiopathic and drug-related groups. In the pa-

tients with an underlying disease such as carcinoma,hepatitis, etc., the Clq and mRFassays were positivein equal frequency.

At least some forms of lymphocytic vasculitis anddiseases exhibiting lymphocytic perivascular infiltratesmay be immune complex-mediated because 41% ofthe serum samples had elevated levels when testedby the mRFassay. Interestingly, only 4 of 48 of thesesamples showed elevated Clq BA; 3 of these sera were

minimally elevated. The ability of an immune complexto activate complement and presumably to interact withClq is dependent upon its size (29, 37). Preliminarydata from this laboratory using Agg IgG indicates thatboth the Clq and mRFassays can detect complexes>19S as well as complexes of intermediate size. ThemRFassay does appear to be the more sensitive of thetwo tests for detection of lighter (7-19S) complexes,whereas the Clq assay is more sensitive in detectingcomplexes >19S. Separation studies of serum fromClq-negative mRF-positive patients revealed the pres-

ence of light mRFreactive material in some anid heavymRF reactive material in others. Thus, the absenceof Clq BA in the sera of these patients must be ex-

plained by more than the size of the immune complexalone.

In a recently published collaborative study com-

paring multiple methods for immune complex detec-tion, 17 patients with leukocytoclastic vasculitis were

included (38). 76% of these sera were reactive by theClq binding assay and 50% were reactive using

the solid-phase mRFassay (27). Our mRFdata (55%reactivity) shows close correlation with this study. Al-though we detected Clq reactive material in a lowerfrequency (54% reactivity), our data is in general agree-

ment with World Health Organization (WHO) results.Interestingly, the soluble mRF assay (28) was lesssensitive (35 vs. 50%) in detecting immune complexesin vasculitis in the WHOstudy when compared withthe solid-phase mRFassay (27). In testing a small seriesof our patients with leukocytoclastic vasculitis, how-ever, both mRFassays appeared to be equally sensitive.

Blood vessel IF was detected in 89% of the biopsyspecimens. Direct IF was positive in approximatelyequal frequency in the two groups of patients (group1, 91% and group 2, 88%). The severity of the reactionand the age of the lesion may be contributing factorsin the frequency of detection of blood vessel IF. Ourdata suggest that direct IF cannot be used to predictthe presence or absence of circulating immune com-

plexes in patients with leukocytoclastic vasculitis,lymphocytic vasculitis, or patients with a lymphocyticperivascular infiltrate.

Total hemolytic complement was depressed in 14of the 71 cutaneous vasculitis patients tested; all but2 of these patients had vasculitis of the leukocytoclastictype. Cryoglobulins were detected in 17 of 70 pa-

tients tested (24%); 15 of these patients had a leuko-cytoclastic type of vasculitis. In all but three cases thepresence of either a depressed CH,, or cryoglobuline-mia was associated with detectable circulating immunecomplexes. The sera of 11 patients with cryoglobuline-mia and 9 patients with depressed CH,. contained bothClq and mRFreactive material. Three patients withcryoglobulinemia and four patients with depressed

1658 S. E. Mackel, G. Tappeiner, H. Brumfield, and R. E. Jordon

b

f~nRF'

56

CH50 had only Clq reactive material. No patient withCH,0 and only one patient with cryoglobulinemia hadcirculating immune complexes detectable by only themRFassay.

By using density gradient ultracentrifugation and gelfiltration chromatography, the Clq and mRFreactivematerial and the anticomplementary material of mostsera eluted in high molecular weight fractions, a sizeconsistent with antigen-antibody complexes. In addi-tion, in one case, we were able to identify a two- tothreefold elevation of HBSAg in these same high mo-lecular weight fractions by radioassay when comparedwith the other fractions. Others have identified HB,Agin cryoprecipitates (39-41), and in cutaneous bloodvessels (39) by direct IF in patients with leukocyto-clastic vasculitis. To our knowledge, this is the firsttime this antigen has been located in high molecularweight fractions, or those that should contain immunecomplexes. Unfortunately, antigen identification invarious forms of cutaneous vasculitis is complicatedby the multiplicity of antigens that may play a role inthe pathogenesis of this disease. Further studies of thistype, i.e., identifying responsible antigens, however,are currently underway.

In 1976 Sams and co-workers (1) proposed a hypo-thetical model for the development of lesions of leuko-cytoclastic vasculitis. This was based upon the deposi-tion of soluble immune complexes in blood vesselwalls, subsequent activation of complement with re-lease of neutrophil chemotactic factors, fixation of neu-trophils to sites of immune complex deposition, andresulting immunologic injury from the release of lyso-somal enzymes. The demonstration of circulating im-mune complexes in a high percentage of patients withactive disease adds further support to their contentionthat leukocytoclastic vasculitis is indeed an immunecomplex disease. Questions such as the antigen in-volved, the role of the reticuloendothelial system inhandling immune complexes, and the importance ofthe complement system in mediating vessel destruc-tion remain to be answered. In addition, the earlychanges taking place that mediate the deposition ofimmune complexes in blood vessel walls remain un-clear. The importance of using more than one type ofassay for the detection of circulating immune com-plexes has also been further substantiated.

ACKNOWLEDGMENTS

Mrs. Jane Kahl provided excellent technical assistance.This work was supported in part by research grant AI 14550

from the National Institutes of Health, U. S. Public HealthService, and by the Medical Research Service of the VeteransAdministration.

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1660 S. E. Mackel, G. Tappeiner, H. Brumfield, and R. E. Jordon