Humoral immune parameters like total immunoglobulins and specific antibody

levels in serum were studied in filarial chyluria patients. Mean serum IgG was significantly

reduced in this group compared to normal controls, while IgA and IgM levels remained

comparable to controls. Anti-filarial antibody titre as measured by enzyme-linked immunosorbent

assay also was significantly reduced. However, the total and specific IgE

antibody titre was similar to that of controls. Specific IgE contents of the patients’ sera could be

related to their microfilaraemic status

A. Date

A. Raja

A. Tandon

A. Voller

Alamelu Raja

D. Rowe

J. Takahashi

K. J. Turner

K. Mehta

K. Saha

O. H. Lowry

P. R. Narayanan

R. G. Hamilton

R. Hussain

R. Prabhakar

R. S. Desowitz

R. S. Paranjape

Rabia Hussain

S. Dissanayake

W. Strober

Journal of Biosciences

Abstract. Humoral immune parameters like total immunoglobulins and specific antibody levels in serum were studied in filarial chyluria patients. Mean serum IgG was significantly reduced in this group compared to normal controls, while IgA and IgM levels remained comparable to controls. Anti-filarial antibody titre as measured by enzyme-linked im-munosorbent assay also was significantly reduced. However, the total and specific IgE antibody titre was similar to that of controls. Specific IgE contents of the patients &apos; sera could be related to their microfilaraemic status

CiteSeerX

Humoral immune response to filarial antigens in chyluria

Raja, Alamelu

Hussai, Rabia

Prabhakar, R

Narayanan, P R

NIRT Institutional Repository

J. Biosci., Vol. 6, Number 5, December 1984, pp. 723-728. © Printed in India.Humoral immune response to filarial antigens in chyluriaALAMELU RAJA, RABIA HUSSAIN*, R. PRABHAKAR andP. R. NARAYANAN†Tuberculosis Research Centre, Madras 600031, India* Laboratory of Parasitic Diseases, National Institute of allergy and Infectious Diseases,National Institute of Health, Bethesda, Maryland 20205, USAMS received 28 August 1984Abstract. Humoral immune parameters like total immunoglobulins and specific antibodylevels in serum were studied in filarial chyluria patients. Mean serum IgG was significantlyreduced in this group compared to normal controls, while IgA and IgM levels remainedcomparable to controls. Anti-filarial antibody titre as measured by enzyme-linked im-munosorbent assay also was significantly reduced. However, the total and specific IgEantibody titre was similar to that of controls. Specific IgE contents of the patients’ sera could berelated to their microfilaraemic status.Keywords. Chyluria, humoral immunity; IgE.IntroductionChyluria is one of the many clinical manifestations of filariasis. It is the commonest ofthe chylous complications of lymphatic obstruction. Several studies in the past haveanalysed the cellular and humoral immune parameters of lymphatic filariasis patients(Desowitz et al., 1976; Paranjape, 1981). Until recently, there have been very fewpublished reports on the status of cellular and humoral immunity in patients withchyluria. We have in our previous experiments observed a reduction in general cellularimmune response (Raja et l., 1983). The present paper summarises our findingsregarding the total and specific immunoglobulin levels and reaginic antibody responsein these patients.Materials and methodsPatients for the study were chosen from those attending the Filariasis Clinic ofGovernment General Hospital, Madras. All the patients had the complaint of passingmilky urine. Five of the patients had circulating microfilaria in their blood, 6 of the† To whom correspondence should be addressed.Abbreviations used: SRID, Single radial immunodiffusion; ELISA, enzyme linked immunosorbent assay;PRIST, paper radio immunosorbent test; DiA, Dirof laria immitis; BmA, Brugia malayi; SeA, Setaria digitata;RAST, radio allergo sorbent test.723724 Alamelu Raja et al.patients had other clinical manifestations of filariasis like lymphadenitis, lymphangitisand hydrocele and 7 did not have any evidence of filariasis. The male : female ratio wasapproximately 5 : 1 and they were spread over a broad age group ranging from 18 yrs to50 yrs. Lymphangiography was done for all the patients. Dilated tortuous lymphaticsand retrograde flow at several sites were observable.Total protein estimationTotal protein estimation in serum was done according to Lowry et al.(1951).Immunoglobulin estimationIgG, IgA and IgM in serum were estimated by single radial immuno diffusion (SRID)following Rowe t al. (1970). Serum IgE was estimated by paper radio-immunosorbenttest (PRIST). (Kit from Pharmacia Diagnostics, New Jersey, USA).Specific antibody measurementSpecific antihehninthic antibodies in serum of class IgG were measured by enzymelinked immunosorbent assay (ELISA). Helminthic antigens Dirofilaria immitis (DiA),Brugia malayi (BmA), and Setaria digitata (SeA) were used. DiA and BmA were salineextracts of the adult worms obtained from the Laboratory of Parasitic Diseases,NIAID, NIH, USA. SeA was an extract of the adult worms in tissue culture mediumRPMI 1640.The sera from normals and patients were tested at a dilution of 1/800. The assay wasdone according to the method of Voller et a . (1976) with slight modifications asreported elsewhere (Narayanan et al.,1983).Specific IgE antibodies were measured by radio-allergo sorbent test (RAST) usingBmA as the antigen (Hamilton et al., 1981).Immunoglobulins G, A and M in 17 chyluria sera have been measured using SRID.Twenty eight normal sera were also included as controls. The results are presented intable 1. Total proteins, IgA and IgM, all remained comparable in chyluria and normalcontrol sera. The mean IgG levels in the serum of chyluria patients was 790± 446 mg/dl, whereas that of normal subjects was 1295 ± 414 mg/dl. This reduction inthe IgG levels was statistically significant (P < 0.001).Anti-helminthic IgG antibody levels were estimated using ELISA. Figure 1 shows themean antibody titres in the patients’ sera and that of endemic controls using BmAantigen. The mean antibody levels in the patient group was lower than the endemiccontrols. Similar reduction was seen also with heterologous antigens of DiA and SeA(figure 2).Serum IgE levels have been estimated using PRIST in 15 patients and 10 normalsubjects. Geometric mean of total IgE level in the patient group was approximately 10times higher than the normal control group. BmA specific IgE levels in the patients asmeasured by RAST were comparable to the normal control sera with mean values ofImmunoglobulins in chyluria 7251 837 195 1772 804 446 1633 609 428 834 609 184 1635 364 148 556 773 180 767 185 267 1508 287 850 559 520 108 4210 358 37 13811 499 50 37612 1803 286 14213 1484 213 16514 1377 203 21315 1097 66 19016 914 85 22517 914 96 169Mean ± S.D.Mean ± S.D.NHS (28)790 ± 4461295 ± 414226 ± 199172 ± 72151 ± 80192 ± 95t 3.85 1.29 1.46p < 0.001 > 0.20 ~ 0.20Figure 1. Anti BmA antibody levels of chyluria patients and control subjects.54 ng/ml for patients and 77 ng/ml for controls. The level of specific IgE was less inmicrofilaraemic patients than the microfilaria negative group (table 2).DiscussionTotal serum IgG levels in chyluria patients was significantly lower than that of normalsubjects. The total serum proteins, IgM and IgA levels of chyluria sera were within the726 Alamelu Raja et al.Figure 2. Levels of antifilarial antibody to heterologous antigens in chyluria sera.Antibody levels have been compared at a dilution of 1/1600 of the sera. Hatched arearepresents the mean ± SD. of the normal sera.Table 2. Total and specific IgE in chyluria sera.Total IgE BmA specific IgES. No. ng/ml ng/ml1* 2880 272 2400 583* 2016 804 5760 525 9120 436 3240 287* 2400 808 3024 149* 8640 6510† 8160 6211† 7680 6612 3366 6613 11040 5514 18720 25415 5640 57GM for patients 5057 54GM for 10 controls 636 77GM for mf +ve group 3780 37GM for mf -ve group 6620 73* Microfilaria positive patients.† Microfilariamic status could not he ascertained.normal range. Many investigators have reported, either an increase in the IgG level ofsera of filarial patients (Desowitz et al.,1976; Saha et al., 1979) or normal level of IgG(Paranjape, 1981). In our patients, the mean serum IgG was significantly reduced thanin normals. But out of the 17 patients studied, only 5 showed drastic reduction in IgG,whereas the others remained within the normal limits. Date et al. (1981) had alsoreported normal IgG levels in chyluria patients. The reduction in the level of IgGobserved in some patients could be due to the loss of proteins from retroperitoniallymphatics into the urinary tract occurring as a result of lymphatic urinary tractcommunication. This was evidenced by the lymphangiograms. Similar bulk loss ofimmunoglobulins into the gastro-intestinal tract and resultant hypogammaglobu-Immunoglobulins in chyluria 727hnaemia in serum was encountered in patients with intestinal lymphangiectasia(Strober et al., 1967). In 4 of the chyluria patients’ urine analysed, considerable quantityof IgG and other proteins were detected, strengthening the possibility that the leakagemay be the main causative factor for hypogammaglobulinaemia of serum. However,additional factors like decreased synthesis or increased catabolism may also play a role.Nonavailability of adult Wuchereria bancrofti for antigen preparation, necessitatedthe use of other antigens prepared from human (BmA) and non-human worms (DiA andSeA) which have been shown to cross-react extensively with W bancrofti (Takahashiand Sato, 1976; Dissanayake and Ismail, 1980; Tandon et al., 1981). These antigenscould be obtained comparatively easily from animal hosts. Mean antifilarial antibodylevels in general in these patients were significantly reduced in comparison withendemic normal controls. The controls themselves showed detectable level of antibodyactivity because they were also sensitized to the endemic helminthic infections. But theresponse shown by the chyluria patients was far lower than the controls themselves,which was in accordance with the reduced serum IgG levels. However, reaginicsensitization to the filarial parasite was not hampered in chyluria patients. Mean totalIgE was 10 times more than that of the normals and antigen specific IgE remainedsimilar to the normals. Such non-specific potentiation of IgE response has beenreported earlier in other helminthic diseases (Turner t al., 1979). A distinct relationshipcould be discerned between filarial specific IgE and parasitological status of thepatients. Mean BmA specific IgE was 73 ng/ml in the microfilariae negative group and37 ng/ml in the microfilariae positive group.The decreased IgE level in the microfilariamic patients could be due to the absorptionof the specific IgE onto the surface of the microfilariae. Evidence for such an absorptionhas been shown by Mehta et al. (1980), where they have demonstrated the adherence ofspleen cells to the microfilariae in the presence of immune serum and that the activeprinciple in the immune serum responsible for cellular adhesion was IgE. The reductionmay also be due to a specific suppression of production of IgE in the presence ofmicrofilariae. Similar low level of specific IgE had been observed in other non-chyluriamicrofilariaemics also (Hussain et al., 1981).AcknowledgementsThe authors are thankful to Prof. S. Subrahmaniam, Head of the Departmentof Microbiology, Post-Graduate Institute of Basic Medical Sciences, Madras;Dr. V. Kumaraswamy, Tuberculosis Research Centre, Madras and Prof.K. V. Thiruvengadam, Head of the Department of Medicine, Government GeneralHospital, Madras for their interest and encouragement during the study.ReferencesDate, A., John, T. J., Chandy, K. G., Rajagopalan, M. S., Vaska, O. H., Pandey, A. P., and Shastry, I. C. M.(1981) Br. J. Urol., 53, 384.Desowitz, R. S., Berman, S. J., and Puloka, T. (1976) Bull. WHO, 54, 565.Dissanayake, S. and Ismail, M. M. (1980) Bull. WHO, 58, 649.728 Alamelu Raja et al.Hamilton, R. G, Hussain, R., Ottesen, E. A. and Adkinson, N. F. (1981) J Immunol. Methods, 44, 101.Hussain, R., Hamilton, R. G., Kumaraswamy, V., Adkinson, N. F. and Ottesen, E. A. (1981) J. Immunol., 127,1623.Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265.Mehta, K., Sindhu, R. K, Subrahmanyam, D. and Nelson, G. S. (1980) Clin. Exp. Immunol., 41, 107.Narayanan, P. R., Acharyulu, G. S., Krishnamurthy, P. V., Ravoof, A., and Tripathy, S. P. (1983) Indian J.Tuberc., 30, 29.Paranjape, R. S. (1981) Immune Response in Filariasis, Ph.D. Thesis, University of Madras, Madras.Raja, A, Kmuuaswamy, V., Narayanan, P. R. and Tripathy, S. P. (1983) Indian J. Med. Res., 77, 314.Rowe, D., Anderson, S. A., and Arab, B. A. (1970) Bull. WHO, 42, 535.Saha, K., Sarkar, N., Paul, D. N. and Ray, D. (1979) Indian J. Med. Res., 70, 22.Strober, W., Wochner, R. D, Carbone, P. P, and Waldmen, T. A. (1967) J Clin Invest, 46, 1643.Takahashi, J. and Sato, K. (1976) Jpn. J. Exp. Med., 46, 7.Tandon, A., Saxena, R. P., Bhatia, B. and Saxena, K. C. (1981) Indian J. Med. Res., 73(suppl.), 93.Tuma, K. J., Feddemma, L. and Quinn, E. A. (1979) Int. Arch. All. Appl. Immunol., 58, 232.Voller, A., Bidwell, D. E. and Bartlett, A. (1976) Bull. WHO, 53, 551.

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Humoral immune response to filarial antigens in chyluria

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