INTERNATIONAL JOURNAL OF LEPROSY^ Volume 58, Number I

Printed in the U.S.A.

Activation of Complement by CirculatingImmune Complexes Isolated from Leprosy Patients'

Padmawati Tyagi, Vadakkuppattu Devasenapathi Ramanathan,Bhawneshwar K. Girdhar, Kiran Katoch,

Amarjit Singh Bhatia, and Utpal Sengupta 2

Elevated levels of circulating immunecomplexes (CICs) have been reported in al-most all types of leprosy patients (2.9• 13. 16)

and deposition/in situ formation of immunecomplexes (ICs) is thought to be a precipi-tating factor for the development of the er-ythema nodosum leprosum (ENL) type ofreaction in lepromatous (LL) leprosy pa-tients (25 ). Although increased levels of CICshave been reported in all types of leprosypatients, the pathophysiological role of thesecomplexes in leprosy has not been studied.However, Saha and his colleagues (20) haveshown that CICs isolated from leprosy pa-tients were able to activate complement.

Laboratory and animal studies haveshown that ICs formed in in vivo conditionsactivate the complement system throughboth the classical pathway and the alter-native pathway ( 3). Miller and Nussenzweig( 12) have shown that activation of comple-ment leads to solubilization of ICs, and oncefully solubilized, ICs lose their ability to ac-tivate complement further and they aretermed end-stage complexes (1.8,12,23).

A recent report by Ramanathan, et al. ( 17 )has shown that reactional leprosy patientsof the tuberculoid and borderline tubercu-loid (TT/BT) and lepromatous (BL/LL)types have a reduced complement-mediat-ed solubilization (CMS) capacity. The totalcomplement functional activity in these pa-tients, however, was found to be normal.

' Received for publication on 9 June 1989; acceptedfor publication in revised form on 11 October 1989.

2 P. Tyagi, M.Sc., Research Assistant; V. D. Ra-manathan, M.B.B.S., Ph.D., Senior Research Officer;B. K. Girdhar, M.D., Deputy Director; K. Katoch,M.D., Senior Research Officer; A. S. Bhatia, M.Stat.,Senior Research Officer; U. Sengupta, M.V.Sc., Ph.D.,Deputy Director, Central JALMA Institute for Lep-rosy, P.O. Box 31, Taj Ganj, Agra 282001, India. Dr.Ramanathan's present address: Tuberculosis ResearchCentre, Chetput, Madras, India.

Reprint requests to Mrs. Padmawati Tyagi.

Whether reduced CMS ability plays any im-portant role in determining the pathophysi-ological nature of CICs in reactional leprosypatients is not known. Furthermore, wheth-er CICs in reactional and nonreactional lep-rosy patients differ in their immunopatho-logical properties is also not known. Sincecomplement activation leads to the gener-ation of a variety of inflammatory reactions,the present study was planned in order tofind out a) whether CICs isolated from thesera of various types of leprosy patients havethe ability to activate complement, and b)whether reactional and nonreactional TT/BT and BL/LL patients differ in their abilityto activate complement pathways.

MATERIALS AND METHODSReagents. Polyethylene glycol (PEG

6000), bovine serum albumin (BSA), andethylene glycol-bis (amino-ethylether) tet-ra-acetic acid (EGTA) were obtained fromSigma Chemical Co., Poole, Dorset, U.K.

Antigen. The cell-free extract (CFE) ofMycobacterium leprae derived from arma-dillo was kindly supplied by Dr. R. J. W.Rees, National Institute for Medical Re-search, London, from the IMMLEP (WHO)Bank.

Antisera. Anti-sheep hemolysin was ob-tained from Span Diagnostic, Surat, India.Anti-human IgG and IgM peroxidase con-jugates were obtained from Sigma ChemicalCo., St. Louis, Missouri, U.S.A.

Buffers and substrate. Phosphate buff-ered saline (PBS, 0.15 M, pH 7.2) and PBScontaining EDTA (0.01 M EDTA, pH 7.6)were used for precipitating CICs from theserum samples. Veronal buffered saline, pH7.35, 0.15 M containing 0.1% gelatin,0.00015 M Ca 2 and 0.005 M Mg 2 +

(GVBS++) was used for the total hemolyticcomplement assay (CHSO). GVBS contain-ing 0.005 M Mg 2+ and 0.05 M EGTA

31

32^ International Journal of Leprosy^ 1990

(GVBSMgEGTA) was used for the alter-native pathway hemolytic complement as-say (AHSO). GVBS containing 0.01 MEDTA (GVBSEDTA) was used for stoppingthe complement reactions. PBS containing0.05% Tween 20 (PBST-20) was used forwashing the ELISA plates. Ten mg ofO-phenylene diamine and 15 pl of 6% H2O,were mixed in 25 ml of 0.1 M citrate phos-phate buffer, pH 5.0, and used as the sub-strate.

Sera. For the isolation of CICs, 10 mlblood samples from each of 80 leprosy pa-tients-20 tuberculoid/borderline tubercu-loid (TT/BT); 20 BT with reaction (I3TR);20 lepromatous (BL/LL); and 20 BL/LL withENL reaction—were collected. The patientswere classified by the criteria of Ridley andJopling ('s). Those patients who gave no his-tory of reactional episodes during the pre-ceding 30 months at the time of blood col-lection were categorized as nonreactionalcases. BL/LL patients who had episodes ofreactions during the preceding months, andthose who were having ENL and TT/13Tpatients in reaction at the time of bloodcollection were designated as reactionalcases. Blood samples collected from 15healthy laboratory volunteers from the Cen-tral JALMA Institute for Leprosy, Agra,were used as control samples. All of the seraobtained from these blood samples werestored at —70°C in 0.5 ml aliquots.

Isolation of CICs by PEG precipitation.CICs were precipitated by 2.5% (w/v) PEG6000 following the method of Creighton, etal. ( 7). The PEG precipitates were freshlyprepared for each experiment from aliquotsof serum samples for each group. One mlofthe aliquots was diluted 1:5 in PBS EDTA.Equal volumes of 5% PEG 6000 in PBSwere added to the diluted sera. After 18-24hr incubation at 4°C, the precipitates wereseparated by centrifugation at 1032 x g x20 min at 10°C. The precipitates werewashed once with 2.5% PEG. They werethen thoroughly dissolved in PBS at 37°Cfor 2 hr, and the volume was reconstitutedto the original serum volume with PBS. AllPEG precipitates were stored at —20°C in0.5 ml aliquots and were thawed once andused in the experiments.

Estimation of total protein content. Totalprotein concentrations in the PEG precip-

itates were estimated using Lowry, et a/.'smethod ('").

Estimation of mycobacterial CICs in PEGprecipitates. The CICs in the PEG precip-itates containing lgG and IgM antibodiesdirected against M. leprae antigens were de-termined using an enzyme-linked immu-nosorbent assay (ELISA) developed in ourlaboratory. The ELISA was carried out asfollows: 50 pl of 100 pg/ml cell-free extractof IL leprac (CFE) was added to each wellof a 96-well microtiter plate (U-bottom;Nunc, Denmark). After overnight incuba-tion at 4°C, the antigen solution was re-moved and the plates were washed twicewith PBST-20. The plates were then incu-bated at 37°C for 2 hr after adding 100 plof 1% BSA in PBS to block all free sites ofeach well. After washing the wells twice withPBST-20, 50 pl of 1/10 diluted PEG pre-cipitates from various types of leprosy pa-tients and normal controls were added tothe wells, and the plates were incubated for2 hr at 37°C. The PEG precipitates were alsoadded to the 1% BSA-PBS-coated wells inaddition to the CFE-coated wells to deter-mine the specific as well as nonspecific bind-ing of the CICs to the I. leprae antigens.After washing the wells with PBST-20 fivetimes, 50 pl of 1:1000 peroxidase-conju-gated antihuman IgG or IgM was added toall the wells, and the plates were incubatedfor 2 hr at 37°C. The plates were finallywashed five times with PBST-20, and 50 plof the substrate was added to each well. Theplates were incubated for 30 min in the dark,and the reaction was stopped by the addi-tion of 50 pl of 7% H 2SO4 . The intensity ofthe final color reaction was read at 492 nmin a Titertek Multiskan plus ELISA reader(Flow Laboratories, U.K.).

Determination of optimal dose of PEGprecipitates for activation of complement.For the classical pathway (CP) of comple-ment activation, 0.025 ml fresh normal hu-man scrum (NHS) was diluted with GVBS*+buffer in the ratio of 1:100. PEG precipitatesand the diluted NHS were mixed in the pro-portions of 1:1, 2:1, 3:1, and 4:1 and wereincubated at 37°C for 1 hr.

For the alternative pathway (AP) of com-plement activation, 0.065 ml NHSMg-EGTA (0.05 ml NHS and 0.005 ml 5 mMMgCl 2 and 0.01 ml 50 mM EGTA) was di-

0.165 ± 0.115(0.00-0.342)

0.566 ± 0.554(0.00-1.86)

0.527 ± 0.200(0.392-0.833)0.670 ± 0.243(0.222-0.843)0.880 ± 0.373(0.183-1.544)

p < 0.05p < 0.05p < 0.001p < 0.001

NSNSNSNSNS

0.035 ± 0.037(0.010-0.034)0.106 ± 0.042(0.043-0.181)0.086 ± 0.074(0.031-0.239)0.343 ± 0.165(0.057-0.584)0.373 ± 0.275(0.060-0.528)

NS'NS

p < 0.001p < 0.001p < 0.001p < 0.001

NSp < 0.001p < 0.001

58, 1^Tyagi, et al.: Complement Activation by CIC^ 33

THE TABLE. Total protein concentrations and 171ycobaderial ill/1111111C complexes (IgGand 101 Oyes) in PEG precipitates jrein sera qf leprosy patient and control groups andtheir ability to consume classical and alternative pathways of complement.a

Group

NHS

TT/I3T

I3TR

BL/LL

ENL

Statistical analysisNHS vs TT/BTNHS vs IITRNHS vs 13L/LLNHS vs ENLTT/BT vs I3L/LLTT/BT vs ENL13T12 vs 13T13 -1'12 vs 13L/LLIITR vs ENL

No Total proteincone. pg/ml

10^82 ± 39(40-140)

9^347 ± 260(80-900)

7^191 ± 89(100-360)

8^331 ± 150(80-620)

8^375 ± 211(120-700)

p < 0.001NS

p < 0.001p < 0.001

NSNSNS

p < 0.05NS

Percentage complementconsumption

CHSO U/ml AHSO U /mI

0.62 ± 1.97 0.87 ± 2.74(0-6.24) (0-8.69)NC' 11.18 ± 2.46

(1.356-33.0)0.83 ± 2.70 15.39 ± 9.48

(0-9.09) (13.8-32.5)7.44 ± 7.34 26.26 ± 16.75(6.09-17.6) (12.7-56.5)4.97 ± 6.99 18.45 ± 14.76

(0-19.2) (5.50-41.3)

NS NSNS p < 0.01

p < 0.05 p < 0.01NS p < 0.01NS NSNS NSNS NSNS NSNS NS

Mycobacterial ICs (OD)

IgG^IgN1

Values are given as mean ± S.D. (range). NHS = normal human sera; TT/BT = tuberculoid/borderline tuber-culoid leprosy; BTR = I3T in reaction; BL/LL = borderline lepromatous/lepromatous leprosy; ENL = BL/LLwith erythema nodosum leprosum.

" NC = no consumption of complement.NS = not significant.

luted with GVBSMgEGTA in the ratio of1:13.5. The PEG precipitates and the di-luted NHS were mixed in the proportionsof 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1, and wereincubated at 37°C for 1 hr.

Determination of CHSO consumption. ThePEG-precipitate-treated NHS samples weresubjected to the residual CHSO assay usingthe method of Mayer ("). Briefly, serial di-lutions of PEG-precipitate-treated NHSwere incubated with 0.1 ml of 2.5% opti-mally sensitized sheep red blood cells(SRBC) at 37°C for 1 hr, followed by theaddition of 2 ml of GVBSEDTA in eachdilution to stop further activation of com-plement. Nonlysed SRBC were spun downat 600 x g x 7 min. The percent hemolysiswas measured in the supernatant for eachdilution at 412 nm in a spectrophotometer(Shimadzu UV-160, Kyoto, Japan).

Determination of AHSO consumption.The PEG-precipitate-treated NHS samples(containing GVBSMgEGTA) were assayed

for the residual AHSO activity using themethod of Platts-Mills and Ishizaka ( 14 ).Briefly, serial dilutions of PEG-precipitate-treated NHS were incubated with 0.05 mlof 2.5% washed rabbit red blood cells(RRBC) at 37°C for 1 hr, followed by theaddition of 1 ml of GVBSEDTA in eachdilution to stop further activation of com-plement. Nonlysed RRBC were spun downat 660 x g x 7 min. The percent hemolysisin the supernatant was measured for eachdilution at 412 nm in a spectrophotometeras above.

Statistical analysis. Group means werecompared by Student's t test. The total he-molytic complement assay (CHSO U/ml)and hemolytic assay for alternative pathway(AHSO U/ml) were calculated by linearregression analysis. Program 6D of the13M DP statistical software (Dixon andBrown, 1987) was used to calculate the cor-relation coefficient and simple linear regres-sion (6).

.40

♦ +^•^4^4^4^4^4^4^♦

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1.4^1.01.2^

1.6^2.0

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900^4

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4

N=^:40R

P1.001

• 4^4^♦^♦^•^♦ •

‘?(K'

P^-R^600^+ R 600

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300^+1 300

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1 11 21

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R=.64 .51 P < .001

1^1

♦

4^4^4^4^+^4^+^4^+

.07^.21^ .4?^.63.14^.2R^.4?^.56^.70

34^ International Journal qf Leprosy^ 1990

IgG OD Value

FIG. I. Correlation between levels of total proteinconcentration (+g/ml) and immune complexes (IgGtype) in PEG precipitates from leprosy patients andnormal controls. Numbers denote the number of iden-tical results.

RESULTSTotal protein content in PEG precipitates.

It can be seen from The Table that the totalprotein concentrations in the PEG precip-itates from TT/BT, BL/LL, and ENL pa-tients were significantly higher than thosein the control group. Among the PEG pre-cipitates from the patient groups, it wasfound that the BL/LL group had a signifi-cantly higher protein concentration than didthe BTR group. However, no marked dif-ferences in protein concentration were ob-served among the TT/BT, BL/LL, and ENLgroups.

Immune complex levels in PEG precipi-tates. The Table shows that the PEG pre-cipitates from the BL/LL and ENL groupshad significantly higher levels of IgG andIgM antimycobacterial antibodies than didthose from the TT/BT, BTR, and controlgroups. The PEG precipitates from TT/BT,BTR, and the control groups had very lowIgM levels. However, the IgG level wasfound to be high in one of the BT patients.No significant differences in the levels ofIgG and IgM types of immune complexes

IgM OD Value

FIG. 2. Correlation between levels of total proteinconcentration (pg/m1) immune complexes (IgM type)in PEG precipitates from leprosy patients and normalcontrols. Numbers denote the number of identical re-sults.

were noticed between the reactional andnonreactional cases of either TT/BT or BL/LL types of leprosy.

Correlation between levels of total pro-teins and mycobacterial immune complexes.A significant positive correlation was foundbetween the values of total proteins and themycobacterial ICs (IgG and IgM type) inthe PEG precipitates. The correlation coef-ficients and the p values obtained were r =0.750, p <0.001 and r= 0.646, p <0.001for IgG and IgM (Figs. 1 and 2).

Dose-dependent effect of PEG precipi-tates on complement. In our initial exper-iments to determine the optimal dose ofPEG precipitates in activating complement,the different concentrations of PEG precip-itates obtained from only the BL/LL groupswere tested for their ability to activate theclassical and alternative pathways. Figure3a shows that while PEG precipitates fromBL/LL sera were capable of activating theclassical pathway at twofold concentrationand above, the PEG precipitates from NHScould do so only at fourfold concentration.As for activating the alternative pathway,Figure 3b shows that while the PEG pre-

b • L L

4:1 5:1

Uaa

1:1^2:1^3:1CONCENTRATION OF PEG PRECIPITATE

50

01-=a 402J(f)

o 30U

Z 2WUa

a 10

a

• LL

NHS

58, 1^Tyagi, et al.: Complement Activation by CIC^35

1.1^21^11^4:1

CONCENTRATION OF PEG PRECIPITATE

FIG. 3. a = Normal human serum (NHS) (25 pl) diluted to 1:100 in GVBS+ 4 was incubated at 37°C for 1hr with 25 pl, 50 pl, 75 p1, and 100 Al of PEG precipitates from NHS and LL sera. The effects of differentconcentrations of PEG precipitates from NHS (0 0) and LL (L L) sera on the classical pathway ofcomplement are shown. b = NHS (50 Al), containing 5 pl 5 mM MgCI, and 10 Al 50 mM EGTA, diluted to1:13.5 with GV13SNIgEGTA was incubated at 37°C for 1 hr with 50 pl, 100 pl, 150 pl, 200 Al, 250 pl, and 300Al of PEG precipitates from NHS and LL sera. The effects of different doses of PEG precipitates obtained fromNHS (0 0) and LL (x—x) sera on the alternative pathway of complement are shown.

cipitates from NHS failed to activate thispathway at any concentration, PEG precip-itates from the I3L/LL sera could activatethe alternative pathway at all concentra-tions. Hence, for subsequent experimentsfor a complement consumption study, weused the PEG precipitates from the variousgroups of subjects at a twofold concentra-tion for CHSO and at an equal (1:1) con-centration for AHSO.

CI-150 consumption. As can be seen inThe Table, although the PEG precipitatesfrom BL/LL patients can activate the clas-sical pathway of complement significantly,PEG precipitates from the TT/BT and BTRpatients and the control group were foundto be inefficient activators of this pathway.The percentage complement consumptionpatterns obtained were: TT/BT, 0%; BTR,0.83% ± 2.70; BL/LL, 7.44% ± 7.34; andENL, 4.97% ± 6.99. PEG precipitates fromone NHS were also found to activate theclassical pathway (6.24%). The difference inthe mean values of percentage consumptionbetween NHS and BL/LL was found to bestatistically significant (p < 0.05); whereasthe differences between NHS and BTR orENL were not found to be statistically sig-nificant (p > 0.1; p > 0.05). No direct he-molytic effect of the PEG precipitates on thesensitized SRBC under similar test condi-tions was observed.

AHSO consumption. From The Table itcan be seen that the PEG precipitates fromall of the leprosy groups were found to ac-tivate the alternative pathway of comple-ment efficiently. The percentage consump-tion patterns obtained were: TT/BT, 1 1.18%± 2.46; BTR, 15.39% ± 9.48; BL/LL,26.26% ± 16.75; and ENL, 18.45% ± 14.76.While the differences in the percentage con-sumption between the control and individ-ual leprosy groups were statistically signif-icant (i.e., NHS vs BTR, BL/LL, and ENL= p < 0.01, 0.01, and 0.01), the differenceswithin the leprosy groups were not found tobe statistically significant (p > 0.05). ThePEG precipitate from one normal serum wasalso found to activate the alternative path-way to a small extent (8.69%).

DISCUSSIONIt is now well established that all types of

leprosy patients have elevated levels of CICs,and this is more evident in BL/LL and ENLpatients (2. 13. IS, 22, 25 , .) The immunochemi-cal analysis of these CICs isolated in theform of PEG precipitates showed the pres-ence of IgG, IgM, IgA, Clq, C3, C4, C-re-active protein, and rheumatoid factor( 5 . ' 6 ). Recently, Ramanathan, et al. ( 17 ) andChakrabarty, et al. (4) have further reporteddiminished complement-mediated solubil-ization of in vitro formed immune com-

36^ International Journal of Leprosy^ 1990

plexes in the sera of13TR, BL/LL, and ENLpatients. Whether the reduced complement-mediated solubilization capacity of reac-tional and nonreactional patients' sera ac-tually renders the CICs unsolubilized inthese patients, and therefore causes harmfuleffects, is not known.

This study is an attempt to find outwhether or not immune complexes in thecirculation of different types of leprosy pa-tients are different in their ability to activatethe complement system in order to betterunderstand the role of immune complexesin leprosy pathology. In the present study,a positive correlation between the proteincontents and the levels of immune com-plexes might indicate (Figs. 1 and 2) thatthe high levels of total protein in the PEGprecipitates are due to an increase in im-mune complex levels.

Saha, et al. ( 20) have demonstrated thecomplement-activating ability in PEG pre-cipitates from leprosy patients for the clas-sical pathway. Like Saha, et al. ( 20), we alsocould not demonstrate any difference in thecomplement-activating ability of the PEGprecipitates obtained from reactional andnonreactional patients. However, their studyincluded only LL and ENL types of leprosy.Our results show that while the PEG pre-cipitates from sera of BL/LL and ENL pa-tients were capable of activating both theclassical and the alternative pathways ofcomplement, PEG precipitates from TT/BTand BTR sera were found to activate thealternative pathway more efficiently than theclassical pathway of complement. This isprobably due to the fact that CICs isolatedas PEG precipitates were found to containmycobacteria ( 19), and mycobacterial com-ponents arc known to activate the alterna-tive pathway of complement 21 ).

The differences in the total protein con-centration in the PEG precipitates from TT/BT, BL/LL, and ENL patients were notfound to be statistically significant. The factthat the total protein concentration in thePEG precipitates from TT/BT patients wasfound to be as high as that of the PEG pre-cipitate from ENL sera is very difficult toexplain. However, it is known that BT lep-rosy is a very heterogeneous group in whichit is common to find a large variation inantibody levels ( 24). The present study alsorevealed a wide range of protein levels in

the PEG precipitates in this group. Probablydue to this range of variation in protein val-ues in PEG precipitates, there is no statis-tical difference in the protein values be-tween the TT/BT and ENL groups.

The differences in the IgG CIC levels inthe PEG precipitates from TT/I3T, BL/LL,and ENL sera were not found to be statis-tically significant. However, the comple-ment activating ability through both theclassical and the alternative pathways wasfound to be much higher in the PEG pre-cipitates from the BL/LL and ENL groupsthan in the PEG precipitates from TT/BTand BTR sera; the reason for this could bethe presence of high levels of mycobacterialIgM types of immune complexes in thesepatients (The Table). Further, in all of theleprosy and control groups, a positive cor-relation was found between the immunecomplex levels in the PEG precipitates andtheir ability to activate the classical and thealternative pathways of complement (Figs.4 and 5). It was interesting to note that whilethe normal human serum PEG precipitateswere unable to activate complement up toa threefold concentration for the classicalpathway and up to a sixfold concentrationfor the alternative pathway, PEG precipi-tates from the BL/LL patients were foundto activate the classical pathway at a twofoldconcentration and above, and the alterna-tive pathway at all concentrations. Thismight indicate that the CICs were not com-pletely solubilized and therefore were avail-able in the BL/LL patients for further ac-tivation of the complement system.

We can conclude from this study that cir-culating immune complexes isolated fromTT/BT and BL/LL leprosy sera were func-tionally different from each other with re-gard to their complement activating ability.However, no significant functional differ-ence was noticed between the PEG precip-itates from reactional and nonreactionaltypes of TT/BT and BL/LL leprosy groups.

SUMMARYCirculating immune complexes isolated

from different types of leprosy sera as poly-ethylene glycol (PEG) precipitates werefound to be efficient activators of the alter-native pathway of complement. PEG pre-cipitates from BL/LL leprosy patients andthose with erythema nodosum leprosum

58, 1^Tyagi, et al.: Complement Activation by CIC^37

4

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4 +^ +^4.

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.07^.21^.35.14^.28

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(IgG+IgM) OD Value

FIG. 4. Correlation between levels of mycobacterialimmune complexes (IgG + IgM) in PEG precipitatesand their classical pathway complement (CHSO U/ml)consumption capacity. Y axis represents percentage(CHSO U/ml) consumption; X axis denotes combined(0D4+2) value for (IgG + IgM).

(19G+IgM) OD Value

FIG. 5. Correlation between levels of mycobacterialimmune complexes (IgG + IgM) in PEG precipitatesand their alternative pathway complement (AHSOU/ml) consumption capacity. Y axis represents per-centage (AHSO U/ml) consumption; X axis denotescombined (0D 49 ,) value for (IgG + IgM).

were found to activate both the classicalpathway and the alternative pathway ofcomplement efficiently, while PEG precip-itates from TT/BT leprosy patients and bor-derline tuberculoid patients in reaction werefound to active the alternative pathway ofcomplement but not the classical pathway.No significant differences were observed be-tween the PEG precipitates from reactionaland nonreactional TT/BT and BL/LL pa-tients in their complement activating abil-ity.

RESUMENSc eoncontrO que los complejos inmuncs circulantes

aislados del suero de diferentes tipos de lepra por pre-cipitaciOn con polietilen glicol (PEG), fueron cficientesactivadores de la via alterna del complemento. Losprecipitados-PEG de pacientes con lepra BL/LL y depacientes con eritema nodoso leproso activaron efi-cientemente tanto la via clasica como la via alterna delcomplemento. Los precipitados-PEG de pacientes conlepra TT/BT y BT en reacciOn, activaron solo la viaalterna pero no la via clasica del complemento. No seobservaron diferencias significativas entre los preci-pitados-PEG de pacientes TT/BT y BL/LL reaccio-

nales y no reaccionales, en su capacidad de activar alcomplemento.

RESUME

On a observe que les complexes immuns circulantsisolês sous la forme de precipitat de polyethilene glycol(PEG) provenant de malades atteints de differentesformes de lepre, agissaicnt comme des activateurs ef-ficaces des voles metaboliques alternatives du comple-ment. Les precipitats PEG recucillis a partir de maladessouth-ant des formes BL/LL de lepre ou bien de sujetsatteints d'erytheme noueux lepreux, activaient effica-cement a la fois la voic metabolique classique et la votealternative du complement. Par contre les prêcipitatsPEG provenant de sujets TT/BT ou de malades atteintsde lepre tuberculcale dimorphe en reaction, n'acti-vaient que la voic metabolique alternative du comple-ment; ils n'avaient pas d'action sur la voie classique.Aucune difference significative n'a Ote constatee entreles precipitats PEG provenant de malades TT/BT ouBL/LL, en reaction ou sans reaction, quant ]cur ca-pacite d'activer le complement.

Acknowledgments. We thank Mr. P. N. Sharma,Mr. C. Pal, Mr. Kuldeep K. Kulshreshta, and Mr. Ma-likhan Singh for their technical assistance. We alsothank Mr. Anil Kumar Chopra for secretarial help and

38^ International Journal of Leprosy^ 1990

Mr. Hari Om for photographic help. We gratefully ac-knowledge the help of Dr. 11. Srinivasan, Director ofthe Institute, in reviewing the manuscript.

REFERENCES1. BAATRUP, G., PETERSEN, I., JENSEMERS, I. C. and

SVEHAG, S. E. Reduced complement mediatedimmune complex solubilizing capacity and thepresence of incompletely solubilized immunecomplexes in sera. Clin. Exp. Immunol. 54 (1983)439-447.

2. WORVANTN, B., BARNETsoN, R. St. C., KRONVALL,

G. K., ZUBLER, R. H. and LAMBERT, P. H. Im-mune complexes and complement hypercatabo-lism in patients with leprosy. Clin. Exp. Immunol.26 (1976) 388-396.

3. CASALI, P., PERRIN, L. H. and LAMBERT, P. H.Immune complexes and tissue injury. In: Inzinu-iwlogical Aspects of Infectious Diseases. Dick, G.,ed. Lancaster, England: Falcon House, 1979, pp.295-342.

4. CHAKRABARTY, A. K., KASHYAP, A., SEHGAL, V.N. and SAHA, K. Solubilization of preformed im-mune complexes in sera of patients with typeand type 2 lepra reactions. Int. J. Lcpr. 56 (1988)559-565.

5. CHAKRABARTY, A. K., MAIRE, M., SAHA, K. andLAMBERT, P. H. Identification of components ofIC purified from human sera. II. Demonstrationof mycobacterial antigens in immune complexesisolated from sera of lepromatous patients. Clin.Exp. Immunol. 51 (1983) 225-231.

6. CHASEN, S. P6D bivariate (scatter) plots. In: BM DPStatistical Software. Dixon, W. J. and Brown, M.B., eds. Berkeley: University of California Press,1987, pp. 133-141.

7. CREIGHTON, W. D., LAMBERT, P. H. and MIE-

SCHER, P. A. Detection of antibodies and solubleantigen complexes by precipitation with polyeth-ylene glycol. J. Immunol. 111 (1973) 1219-1227.

8. CZOP, J. and NUSSENZWEIG, V. A. Studies on themechanism of solubilization of immune precipi-tates by scrum. J. Exp. Med. 143 (1976) 615-630.

9. GELBER, R. H., DRUTZ, D. J., EPSTEIN, W. V. andFASAL, P. Clinical correlates of C l q, precipitatingsubstances in the sera of patients with leprosy. Am.J. Trop. Med. Hyg. 23 (1974) 471-475.

10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.and RANDALL, R. J. Protein measurement withthe Folin-phenol reagent. J. Biol. Chem. 193 (1951)265-275.

11. MAYER, M. M. Complement and complement fix-ation. In: Experimental immunochemistry. 2nd ed.Kabat, E. A. and Mayer, M. M., eds. Springfield,Illinois: Charles C Thomas, 1961, pp. 133-240.

12. MILLER, G. W. and NUSSENZWEIG, V. A new com-

plement function: solubilization of antibody-an-

tigen aggregates. Proc. Natl. Acad. Sci. U.S.A. 72(1975) 418-422.

13. MORAN, C. J., RYDER, G., TURK, J. L. and WAT-

ERS, M. F. Evidence for circulating immune com-plexes in lepromatous leprosy. Lancet 2 (1972)572-573.

14. PLArrs-MILLS, T. A. E. and ISHIZAKA, K. Acti-vation of the alternate pathway of human com-plement by rabbit cells. J. Immunol. 113 (1974)348-358.

15. RAMANATHAN, V. D., CURTIS, J. and TURK, J. L.Activation of the alternative pathway of comple-ment by mycobacteria and cord factor. Infect. Im-mun. 29 (1980) 30-35.

16. RAMANATHAN, V. D., PRAKASH, 0., RAMU, G.,PARKER, D., CURTIS, J., SENGUPTA, U. and TURK,

J. L. Isolation and analysis of circulating immunecomplexes in leprosy. Clin. Immunol. Immuno-pathol. 32 (1984) 261-268.

17. RAMANATHAN, V. D., SHARMA, P., RAMU, G. andSENGUPTA, U. Reduced complement mediatedimmune complex solubilization in leprosy pa-tients. Clin. Exp. Immunol. 60 (1985) 533-558.

18. RIDLEY, D. S. and JOPLING, W. H. Classificationof leprosy according to immunity; a five-groupsystem. Int. J. Lepr. 34 (1966) 255-273.

19. SAIIA, K., CHAKRABARTY, A. K. and PRAKASH, N.A quick method of demonstrating bacillaemea inpatients with lepromatous leprosy and ultrastruc-tural studies of the circulating acid fast bacilli.Trans. R. Soc. Trop. Med. Hyg. 77 (1983) 660-664.

20. SAHA, K., CHAKRABARTY, A., SHARMA, V. K. andSEHGAL, V. N. Polyethylene glycol precipitates inserum during and after erythema nodosum lepro-sum -study of their composition and anticomple-mentary activity. Int. J. Lepr. 52 (1984) 44-48.

21. SAHA, K., SHARMA, V., CHAKRABARTY, A. K. andSEHGAL, V. N. Breakdown product of factor B asan index of complement activation in lepromatousleprosy and its relation with bacillary load. Scand.J. Immunol. 17 (1983) 37-43.

22. SHWE, T. Immune complexes in glomeruli of pa-tients with leprosy. Lcpr. Rev. 42 (1972) 282-289.

23. TAKAHASHI, M., TAKAHASHI, S. and HIROSE, S.

Solubilization of antigen-antibody complexes: anew function of complement as a regulator of im-mune reactions. Prog. Allergy 27 (1980) 134-166.

24. TOUW LANGENDUK, E. J. M., WARNDORF VAN

DIEPEN, T., HARBOE, M. and BELEHU, A. Relationbetween anti-Mycobacterium leprae antibody ac-tivity and clinical features in borderline tubercu-loid (BT) leprosy. Int. J. Lcpr. 51 (1983) 305-311.

25. WEMAMBU, S. N. C., TURK, J. L., WATERS, M. F.R. and REES, R. J. W. Erythema nodosum lepro-sum a clinical manifestation of the Arthus phe-nomenon. Lancet 2 (1969) 933-935.