Humoral immune response during filarial fever in Bancroftian filariasis

230

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1993) 87, 23&233

Humoral

immune

response

during filarial fever in Bancroftian

S. K. Kar*, J. Mania and P. K. Kar

Clinical Division, Regional Medical Chandrasekharpur, Nandan Kanan Road, Bhubaneswar-751016, Orissa, India

filariasis

Research Centre (ICMR),

P.O.

Abstract Humoral immune responsesagainst filarial parasitic infection were studied in 62 casesof acute filarial diseasepresenting with ‘rilarial fever with adenolymphangitis, in a community where Bancroftian filariasis was endemic, during and about one month before and after the febrile episode. Their total leucocyte and differential peripheral blood cell counts and anti-streptolysin 0 titre were determined and compared. Polymorphonuclear cellular responsesand anti-streptolysin 0 titre did not show any significant alteration during and after fever. Three of 53 previously amicrofilaraemic subjects (9 of whom were initially microfilaraemic) had microfilaria in their circulation during fever, with a significant increasein their geometric mean microfilaria count. Titres of specific immunoglobulin (Ig) G and IgG4 antibody to Wuchereria bancrofti microfilarial excretory/secretory antigens (measuredby enzyme-linked immunosorbent assay)decreasedsignificantly during the fever and the lower levels were maintained one month after fever. The mean circulating immune complex level increased significantly during fever, and a significant percentageof casesdemonstrated circulating filarial antigen during fever, which declined after one month, suggesting the releaseof filarial antigen into the circulation during fever which bound to antibodies to form immune complexes. These observations do not support the suggestion that bacterial infection is the aetiology of filarial fever. It is postulated that antigens releasedfrom parasitesinto the circulation during parturition by adult worms may evoke an allergic responsein the host, causing periodic febrile episodes. Introduction Filarial fever is considered to be a common classical manifestation in both acute and chronic stagesof lymphatic filarial disease. In areas endemic for Bancroftian filariasis, the inflammation may involve the lymph channels of testes, spermatic cord and epididymis besides those of the extremities (WHO, 1984). However, the aetiology of filarial fever is not precisely known. It is sometimes described as a manifestation of secondary infection or local disturbances of lymph flow (MANSON-BAHR & BELL, 1987) or as an allergic manifestation of parasitic infection JOTTESEN. 1984). This studv was initiated to identify any alteration in clinical, parasitological and immunological status of the host during the febrile episode in the natural course of filarial infection. Patients and Methods Sixtv-two established casesof acute filarial diseaseof both sexes(43 males and 19 females)experiencing lilarial fever from a village of Puri district, Orissa, where Bancroftian filariasis is endemic, were included in the study. In a routine survey, basic lilariometric data and serawere collected neriodicallv from patients with filarial disease. Those who manifested filahal fever approximately one month after their initial examination were included in the studv. The criteria used for diagnosis and selection of casesof filarial fever were (i) established acute filarial diseasewith previous history of adenolymphangitis; (ii) no evidence of trauma, injury, septic infection or skin lesion; (iii) evidence of fever accompanying adenolymphangitis affecting limb and scrotum with or without lymphoedema; (iv) no other associatedclinical diseaseexcept the filarial lesion; and (v) no history of antifilarial treatment during at least 6 months preceeding the episode. Clinical caseswith septicaemiacomplicating tilarial infection or with severetoxaemia were excluded. Clinical examinations were carried out during and following the fever episode, and signs or symptoms-wererecorded: No antifilarial medication was riven durine or within one month after the fever. Episodic attack w& controlled by bed rest and antipyretics and antibiotics (cotrimoxazole) in the usual dosage.Antibiotics were used for ethical reasons, as this is standard medical practice in this region for treating acute attacks. Laboratory examination

Microfilarial periodicity testing earlier with 25 known microfilaraemic subjects in the community did not show any significant difference between microfilarial counts in *Author for correspondence.

20 mm3 of blood collected at 21:00 and 23:00 (unpublished observation). Hence, 5 ml blood samples were drawn at 21:30 from each patient during and approximatelv one month after fever (mean 30.3k2.7 d). These were compared with previous blood samples collected approximately one month before the fever attack (mean 29.452.8 d). Of the blood samples, 1 ml (collected with ethylenediaminetetraacetic acid) was used for microfilaria detection and counting by the Nuclepore@membrane filtration technique (3 pm pore size, Nuclepore Corporation, Pleasanton, California, USA), and 1 ml for haematological examination which included differential and total leucocyte counts. The remainder of the blood was allowed to clot, centrifuged, and serum collected and stored in aliquots at -20°C with sodium azide as preservative. Preparation of Wuchereria bancrofti microfilarial excretorylsecretory antigen W. bancrofti microlilariae were separated from blood

by Nuclepore@ membrane filtration (3 un-~)and maintained (3000-4000 per ml) in medium RPMI-1640 buffered with sodium HEPES, supplemented with glutamine and gentamicin (KHARAT et al., 1980). The culture fluid was collected every 24 h and centrifuged at 13 OOOxg. The protein content of the supernatant fluid containing antigen collected over 72 h was estimated by the method of LOWRY et al. (1951). The fluid was preserved with 0.1% sodium azide and stored at -20°C. This antigen (W.b.mf-ES) was used in enzyme-linked immunosorbent assays (ELISAS) to estimate filaria-specific immunoglobulin (1g)Gand IgG4 in patients’ sera. Assessmentof antibody levels by ELISA ELISA was used to measurethe total IgG and IgG4 antibodv levels as described nreviouslv NOLLER et al..

1979). Briefly, polyvinylchloride microtitre plates (Linbro, USA) were coated with W.b.mf-ES at a concentration of 0.15 ug per ml (15 ngiwell) in sodium carbonate-bicarbonate buffer (pH 9.6). The optimum concentration of W.b.mf-ES used for coating the wells was determined by chequerboard titration. Plates were incubated overnight at 4°C drained and washed in 0.01~ phosphate-buffered saline, pH7.2 (PBS) containing 0.5% Tween 20@(PBST). The wells were blocked with 100 ul of PBS containing 3% bovine serum albumin (BSA),‘incubated at 37°C for 1 h and washed 3 times with PBS-T. Two-fold serial dilutions of the test sera (1: 10 to 1: 1280) in PBS-T were dispensed (100 @well) to wells and incubated at 37°C for 2 h. Unbound antibodies were removed by washing

231 the plates 3 times with PBS-T. Peroxidase-conjugated rabbit antihuman IgG (Dakopatts, Denmark) was added at 1:1000 dilution to each well. The optimum dilution of conjugate was determined by chequerboard titration. Plates were incubated for 2 h at 37°C washed 3 times with PBS-T, and 100 ~1of substrate (40 mg ofp-phenylenediamine in 50 ml of distilled water containing 30 ul of 30% H202) were added to each well. Plates were left in the dark for 30 min and the reaction was terminated by addition of 0.05 ml 8N H2SO4 to each well. The optical density was measured at 492 nm with a micro-ELISA reader (Biorad, USA). Since 3 of 14 non-endemic normal European sera gave a positive reaction at 1:160 serum dilution. the omical densitv of the sera at 1:320 dilution was used to determine background absorption. Similar techniques were used to estimate the IgG4 subclass in test sera, including the amount of-coating antigen. After incubation of two-fold serially diluted human sera, the plates were washed and rabbit antihuman IgG4 (Jenssen Biochemica, Belgium), diluted 1:100, was added to each well and incubated for 1 h at 37°C. After 3 washes with PBS-T. swine anti-rabbit Ie conjugated with peroxidase (Dakopatts, Denmark): diluted l:lOOO, was added and incubated overnight at 4C. The bound enzyme activity was then measured as described above. Since 1 of 14 non-endemic European normal seragave a positive reaction at 1:160dilution, the optical density of the sera at 1:320 dilution was used as the background value. Filaria-specific circulating antigen

Circulating filarial antigen was detected by ‘sandwich’ ELISAas described by VOLLERet al. (1976) and modified by REDDY et al. (1984). The optimum dilutions of the first coating of lilaria-specific IgG (FSIgG) and FSIgGpenicillinase conjugate were determined by chequerboard titration. Briefly, 100 ~1of optimally diluted FSIgG (25 ygiml) in carbonate buffer (0.06 M, pH 9.6) were added to each well of a polyvinylchloride microtitre plate (Linbro, USA) and incubated at 37°C for 3 h. After draining the plates were washed in PBS/T. To each well 200 ul of 3% BSA in PBS were added and the ulates were incubated at 37°C for 1 h. After washing the plates 8 times with PBS/T, 100 ~1 of the serum sample diluted 1:300 in PBS/T were added to each well and the plates were incubated at 37°C for 3 h. The plates were washed, 200 ul of 3% BSA in PBS/T were added to each well, and they were incubated at 37°C for 1 h. After rewashing the plate, to each well were added 100 ~1 of FSIgG conjugated to penicillinase (Sigma, USA) diluted 1:400 in PBS/T. The plates were then incubated overnight at 4°C. After further washing with PBS/T, 100 ul of substrate solution were added to each well and the plates were allowed to remain at room temperature for the reaction to occur. The substrate (150 mg of soluble starch in 27.5 ml of 0.25 M sodium phosphate buffer, pH 7.2, containing 10.6 mg- -. penicillin and 100 ul of 0.08 M iodine in 3.2 M potassmm iodine solutionj was prepared freshlv before use. The reaction was terminated after 30 min by adding 25 ~1 of 5N HCI and the results were evaluated visually. Complete decolorization or decolorization with only a slight tinge of substrate colour remaining denoted a positive reaction. Decolorization resulted from the reaction between the iodine in the starch-iodine complex with penicilloic acid produced by the action of the penicillinase on penicillin (NOVICK, 1962). Persistenceof the blue colour indicated a negative result. Since 3 of the 14 non-endemic negative control sera gave a positive result at 1:160 dilution, a serum dilution of 1:300 was used for screening the samples. Circulating immune complexes

The level of circulating immune complex (CIC) was determined by polyethylene glycol (PEG) precipitation

(HASKOVAet al., 1978). Briefly, the serawere diluted 1:3 in borate buffer (0.1 M, pH 8.4). From this, an aliquot of 110 ~1 was further diluted in 1 ml of borate buffer to serve as control. Another aliquot (110 yl) was diluted in 4.166% PEG 6000 in borate buffer and used as the test sample. After thorough mixing the samples were left at room temperature for one hour and then the absorbence was measured at 450 nm. The difference in absorbence F;;y the test and control samples indicated the CIC Anti-streptolysin 0 titre

The anti-streptolysin 0 titre in sera was determined using the kit obtained from Laboratory Diagnostic Company, Inc. (New Jersey, USA), according to the manufacturer’s instructions. In brief, a 5% cell suspension was prepared using fresh rabbit blood in streptolysin 0 buffer. Serum samples were diluted with the streptolysin 0 buffer. A red cell control and a reference standard (supplied with the kit) were always included. Streptolysin 0 reagent was added to all tubes except the red cell control; the-rack with the tubes was shaken gently and incubated at 37°C. A fixed amount of red cell susnension was then added to all tubes, which were again shaken gently and incubated for 30 min. Immediately after incubation, the tubes were shaken again to resuspend cells and examined for haemolvsis. The anti-strentolvsin 0 titre for each serum was measuredas the reciprocal of the highest dilution showing no haemolysis. Statistical analysis

Student’s paired t test was used to compare the eosinophi1 percentages, antibody and CIC levels. The percentagesof seracontaining specific tilarial circulating antigen were compared by means of the z test. Results

The mean febrile period was 2.6tl.O d (range l6 d). The body temperature was 38.2+0$X [100.8+ l.O3”F] (range 37.2-39.6”C [98.9-103.3”F]). Acute lymphangitis affecting lower and upper limbs was observed in 41 cases(65%) and the rest showed scrotal lymphangitis. Parasite count and haematology

Approximately one month preceeding the fever only 9

II

AFTER

DURING

BEFORE

Fig. 1. Geometric mean microfilaria count before, during and after fever (n=9, 12 and 11 respectively); error bars indicate +2 standarderrors.

232

cases(14.5%) had circulating microfilariae (mf) in their peripheral blood (geometric mean count= 103+ 3.04 mf/ml). During the febrile state, 3 more subjects became microfilaraemic (geometric mean=638+ 1.6 mfiml, range 52-5830, n=12). During fever there was a significant rise in microfilarial density, which dropped significantly one month after fever (Fig. 1). Although the mean total leucocyte count and the percentageof polymorphonuclear cells were slightly elevated during the fever episode, they remained within the normal range. There was a significant rise in the percentage of eosinophils during the fever which remained unchanged one month later (Table). Table. Total leucocyte count and percentages of polymorphs and eosinophils before, during and after episodes of Maria1 fever

Cell counta Total leucocytes Polymorphs (%) Eosinophils (%)

Before fever

During fever

After fever

;:‘;x;‘2”3 . . 11.58k6.14

7467 f1744 56.1 k5.8 13*0gbk4.92

7373fl753 55.Ok5.4 12.84k4.8

“Means f standard deviations; n=62. bProbability vs. count before fever
Immune status There was a significant drop in the mean titre of filaria-specific IgG and IgG4 (P
ii 0.00;

BEFORE DURING

Fig. bulin fever from

AFTER

2. Wucherena bancrofti microflarial excretory/secretory immunogloG antibodies and those of subclass IgG? before, during and after in 62 cases of filarial fever (0, IgG: +, IgG4) and 14 normal subjects a non-endemic area (m, IgG; 0, IgG4); means 52 standard errors.

No significant difference was observed between the percentages of caseswith a positive anti-streptolysin 0 titre, during or after the fever. Sera from 35 cases (56.5%) had a positive titre (> 166 Todd units) before the fever; 2 more sera developed a positive titre during fever (59*7%), and one more did so one month after the fever episode (61.3%). There was no significant difference in the percentage of subjects with positive antistreptolysin 0 titres during or after fever in comparison to those before the fever episode. Discussion

Earlier reports have incriminated infections with common bacteria such as streptococcus and/or staphylo-

0.150> I51 ,0.125ZE wp= 0 0. ioo69 OH-

0.075-

E 0 0.050-

I

BEFORE DURING

AFTER

Fig. 3. Mean circulating immune complex level before, during and after fever (n=62); error bars indicate f2 standard errors.

coccusas the causeof episodic filarial fever. Leucocytosis is a common feature of inflammatory reactions, especially those induced by bacterial infections. However, in the present study the mean total leucocyte count in all patients during the fever did not show any significant change, and no significant increase in neutrophil percentagewas observed. The anti-streptolysin 0 titre, an indicator of recent streptococcal infection (WILLIAMS, 1958), was not high in the study caseseither during or after the fever. There was no significant rise in the percentage of caseswith a positive anti-streptolysin 0 titre during or following fever. Hence a bacterial infection as the causeof fever in our subjects can be ruled out. However, the presenceof antibodies to staphylococcal or fungal infections was not investigated. There was a significant rise in the mean microfilarial density and 3 of 53 previously amicrofilaraemic caseshad microtilaria in their peripheral circulation during the febrile episode. This suggestsfresh recruitment of a large number of microfilariae into the circulation during fever, which might have resulted from fresh parturition by one or more adult female(s). The rise in mean eosinophil percentage during the fever was possibly due to the role of thesecells in antigenic clearance. Filaria-specific IgG4 is reported to be a significant marker of infection (OTTESEN et al., 1985). It is suggested that this antibody isotype blocks the binding of IgE and thus leads to allergic responses. During lilarial fever a significant fall in the titre of filaria-specific IgG4 was observed, together with a concomitant fall in the filaria-specific IgG level. A reverse trend was observed in the percentage of cases showing circulating filarial antigen. The mean CIC level also showed a significant rise during fever episodes. These changes indicate that the excess antigen liberated into the circulation might have formed immune complexes after combining with the antibodies. The high level of immune complexes thus produced during fever might have played an important role in the genesisof an allergic responsein the host, resulting in fever by involving macrophages and subsequent releaseof interleukin- 1. It is known that the immune complex is an immunoregulator of the host’s immune response in filariasis. Immune complexes have been shown to mediate a variety of immune suppressive effects on the function of T and B lymphocytes and on the presentation of antigens on the surface of macrophages (SISLEYet al., 1987). The antigens are phagocytosed by macrophages which release endogenous pyrogens chemically similar or identical to interleukin-1. This is important in inducing lymphocyte activation during immune reactions. Macrophages are the major cell type producing endogenous pyrogens, the agents responsible for the induction of the febrile response. Once interleukin-1 synthesis has been initiated by contact of macrophageswith the infectious agent, further produc-

233

tion is maintained

or enhanced by the resulting fever 1984). Hence it appears imperative to undertake further investigations on changes in levels of interleukins and specific immune complexes during fever. In the present study, evidence of acute bacterial infection during fever was conspicuously absent. It can be presumed that the immune-mediated response, through CICs, induces filarial fever and a local inflammatory response such as adenolymphangitis in the host. Parturition fluid released by adult female parasite(s) dwelling in a lymph node of the host, liberated from time to time, may be postulated as being the ‘trigger’ which induces immune-mediated fever episodes. (ASHMAN

& MULLBACHER,

Acknowledgements We thank Dr S. P. Tripathy, Director General of ICMR and the Director, Regional Medical Research Centre, Bhubaneswar for their guidance and encouragement. We also thank Mr S. C. Rout, Mr T. Moharana, Mr K. Dhal and Mr R. N. Nayak of the Clinical Division for their strenuous work in the field during the study.

References Ashman, R. B. & Mullbacher, P. (1984). Infectious diseases, F;le,r and the immune response. Immunology Today, 5, 268Haskova, V., Kaslik, J., Riha, I., Mitl, I. & Rovensky, J. (1978). Simple method of circulating immune complex detection in human sera by polyethylene glycol precipitation. ZeitschriffiirImmunitiits Forschung, 154,399-406. Kharat, I., Satyanarayana? U.,, Ghirnikar, S. N. & Harinath, B. C. (1980). In vitro cultwation of Wuchereria bancrofti microfilariae. Indian Journal of Experimental Biology, 18, 12451247. Lowry, O., Rosebrough, H. & Farr, A. (1951). Protein measurement with the Fohn-phenol reagent. Journal of Biological

Chemistry,103,265-275.

Manson-Bahr, P. E. C. & Bell, D. R. (editors) (1987). Munson’s Tropical Diseases, 19th edition, London: ELBSiBailliere Tindal; pp. 353-406: Novick, R. I’., (1962). Micro iodometric assay for penicillinase. BiochemicalJournal, 83,236-240. Ottensen, E. A. (1984). Immunological aspects of lymphatic filariasis and onchocerciasis in man. Transactions of the Royal Society of Tropical Medicine and Hygiene, 78, supplement, 918. Ottensen, E. A., Skvaril, F., Tripathy, S. I’., Poindexter, R. W. & Hussain, T. (1985). Prominence of IgG4 in the IgG antibody response in human filariasis. Journal of Immunology,

134,2707-2712. Reddy, M. V. R., Malhotra, A. & Harinath, B. C. (1984). Detection of circulating antigen in Bancroftian filariasis by sandwich ELBA using iilarial serum IgG. Journal of Helminthology, S&259-262. Sisley, B. M., Mackenzie, C. D., Steward, M. W., William, J. F., O’Day, J., Luty, A. J. F., Braga, M. & El Sheikh, H. (1987). Association between clinical disease, CIC and Clq binding immune complexes in human onchocerciasis. Parasite Immunology, 9,447-463. Voller, A., Bartlett, A. & Bidwell, D. E. (1976). Enzyme immunoassays for parasitic diseases. Transactionsof the Royal Society of Tropical Medicine and Hygiene, 70,98-106. Voller, A., Bidwell, D. E. & Bartlett, A. (1979). The enzymelinked immunosorbent assay (ELBA). A guide with abstract of microplate application. Alexandria, Virginia, USA: Dynatech Laboratories. WHO (1984). Fourth Report on Lymphatic Filariasis. Geneva: World Health Organization, -Technical Report Series, no. 707. Williams, R. E. 0. (1958). Laboratory diagnosis of streptococcal infection. Bulletin of the World Health Organization, 19, 153.

Received 2 January 1992; revised 2.5June 1992; accepted for publication 26June I992

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