Int. Z lmmunopharmac., Vol. 15, No. 3, pp. 395-400, 1993. Printed in Great Britain.

0192-0561/93 $6.00 + .00 Pergamon Press Ltd.

©1993 International Society for lmmunopharmacology.

C E L L U L A R I M M U N E RESPONSIVENESS IN RABBITS WITH SETARIA DIGITA TA FILARIAL A N T I G E N A N D TDM A D J U V A N T

ALPANA SHARMA and S. N. UPADHYAY

School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi -- 221 005, India

(Received 2 June 1992 and in final form 30 October 1992)

Abstract -- The purified surface antigens of the bovine filarial parasite Setaria digitata were used as an antigen to immunize rabbits. The aqueous suspensions of trehalose 6-6' dimycolate (TDM) has been successfully used as an effective immunomodulator in experimental studies on filariasis. The effectiveness of such an antigen-TDM combination was demonstrated by enhanced humoral and cellular immunity. Administration of antigen alone shows only humoral immunity. The detectable cellular immune responses further confirm the effect of filarial antigen - TDM combination. The cell-mediated immunity was expressed in vivo by delayed skin reaction and in vitro by leukocyte and macrophage migration inhibition tests.

Filariasis causes untold pain, misery and impairment of health like elephantiasis, blindness, hydrocoele and the general illness of millions of people in the world. The human infections generally occur due to Wuchereria bancrofti, Brugia malayi, Loa loa and Onchocerca volvulus. All the filarial parasites are vector borne, transmitted by mosquitoes, biting midges, tabands and black flies. These parasites elicit a broad spectrum of clinical manifestations like asymptomatic microfilaraemia, amicrofilaraemia, chronic infection with lymphatic pathology, endemic controls resisting infection, etc. The study of immune status in filariasis is a little difficult because the immune responsiveness changes in different clinical manifestations.

This paper describes the activity of an aqueous suspension of TDM as adjuvant for immunizing albino rabbits with Setaria digitata sheath antigen for the first time. It also deals with the study of cellular immune responsiveness in rabbits with the combination of S. digitata antigen plus TDM. Since filarial antigens are not completely able to stimulate on their own a strong humoral and cellular immunity, the use of adjuvant in combination with a filarial antigen is the better way to elicit specifically both the immune responses.

TDM a mycobacterial glycolipid is a known immunomodulator. The use of TDM emulsion in oil leads to secondary reactions such as production of granulomas (Ramanathan, Curtis & Turk, 1980) especially after intravenous injections and enhancement of endotoxin lethality, etc. Aqueous

suspensions of TDM can protect mice against infection by Klebsiella pneumoniae and Listeria monocytogenes (Parant et al., 1977), Schistosoma mansoni (Olds, Chedid, Lederer & Mahmoud, 1980), Babesia mieroti (Clark, 1979) and Plasmodium berghei (Kumar, Ahmad & Lederer, 1984). Such suspensions have also been shown to enhance in vitro and in vivo antitumor activity of macrophages (Orbach-Arbouys, Tenu & Petit, 1983). Several workers have reported the adjuvant activity of TDM in mice (Bekierkunst, Levij, Yarkoni, Vilkas & Lederer, 1971; Kumar et al., 1984; Kierszenbaum, Zenian & Wirth, 1984), rats (Saito et al., 1976) and rabbits (Sharma, Haq, Ahmad & Lederer, 1985). Recently a review article (Lemaire, Tenu, Petit & Lederer, 1986) explained more details and mechan- isms of action of TDM as an immunoadjuvant.

E X P E R I M E N T A L P R O C E D U R E S

Antigen

Adult bovine filarial worms (S. dig#am) were collected from a local slaughter house (Gol Kunda, Varanasi) in normal saline. Worms were washed and cleaned in distilled water thrice then exsheathed. The separated sheaths were cleaned and homogenized in a glass homogenizer for 10 rain (five strokes) at 4°C in 10 mM Tris and 20 mM EDTA, pH 5.0. The homogenate was incubated at 37°C for 2 h with intermittant shaking. It was then centrifuged at

395

396 A. SHARMA and S. N. UPADHYAY

Table 1. Weekly immunization schedule

Weekly immunization doses (ml)

Group 1 st 2rid 3rd 4th

I Antigen + TDM 0.25 0.25 No inoculation 0.50 1.00 1.00 in this week 1.00

II Antigen 0.25 0.25 0.50 III TDM 1.00 1.00 1.00 IV Saline 1.50 1.50 1.50

Setaria antigen concentration -- 4 mg/ml. TDM concentration - - 1 mg/ml. Total inoculation in each injection was made to 1.5 ml by addition of saline.

15,000 x g for 20 min. The supernatant was then purified.

Purification

The CNBr activated CL sepharose 4B beads were adsorbed with 33 °70 ammonium sulphate precipitated human filarial gamma globulins from chronic filarial sera. These globulins were coupled to activated sepharose beads according to the method of Kaliraj, Patel, Desai, Megha & Desai (1983) and Gerald Theodore & Kaliraj (1990). The immunoadsorbed sepharose beads were packed in a column and the extracted sheath antigens were passed through it. The non-specific serum proteins were removed by washing the column. The adsorbed antigen was eluted with 3M KCN (pH 6.8). The protein peak concentration was estimated by Lowry's method (Lowry, Rosebrough, Farr & Randall, 1951) and used as a filarial antigen for immunization.

Adjuvant

Trehalose dimycolate was a commercial preparation (Choay-chimie, Montrouge, France) containing 1 mg of TDM per ml of sterile, pyrogen free water.

Animals and immunization schedule

Twenty-eight male, albino rabbits each weighing 1 - 1.2 kg were used for immunization in 4 groups, each containing 7 animals. Group I rabbits were immunized with antigen + TDM, Group II with antigen alone, Group III and Group IV were given antigen and saline only. TDM was administered at the rate of 1 mg/kg body weight. A total of 3 immunizing doses were given - - the first two one week apart and the third on the 4th week. All immunizations were carried out through intravenous route. The details of immunization schedule are given in Table 1.

Indirect hemagglutination test ( I l ia )

Weekly antibody titers were determined by the modified indirect hemagglutination (IHA) technique of Krupp (1969). A 2.5°7o suspension of purified sheep erythrocytes was tanned and sensitized with sheath antigen at 37°C for 20 min. The cells were added to a two-fold dilution of the serum, and the endpoint was taken as the lowest dilution giving 50% cell agglutination. A serum was considered positive if its antibody-titre was higher than 1 : 64.

Enzyme-linked immunosorbent assay (ELISA )

It was performed weekly to detect antibodies in weekly sera samples according to the methods given previously (Sharma et al., 1985). Flat bottomed polystyrene microtitre plates (Laxbro, India) were coated with 0.2 ml of antigen protein (50/ag/ml in 0.05 M carbonate buffer, pH 9.6) in each well and incubated at 4°C for 24 h. The plates were washed 5 times with PBS/T (phosphate buffered saline containing 0.0501o Tween 20, pH 7.4). A 0.2 ml sample of the test serum serially diluted from 1 : 4 to 1 : 65,536 with PBS/T was added to each well and the plates were incubated at 37°C for 30 rain. After incubation, the plates were again washed and 0.2 ml of anti-rabbit immunoglobulin G-alkaline phospha- tase conjugate (A 8025 Sigma; 1 : I000 dilution with PBS/T) was added and incubated for 30 rain at 37°C. Later 0 .2ml of substrate (p-nitrophenyl phosphate, Sigma; 1 tablet for each 5 ml of 10% diethanolamine buffer) was added. The reaction was stopped after 30 rain by adding 0.05 ml of 3 M NaOH and the absorbance was measured at 400 nm.

Skin test

All the rabbits were challenged with 15/ag of antigen protein given intradermally on day 35 according to the methods given previously (David, A1-Askari, Lawrence & Thomas, 1964; Sharma,

Immune Response and a Filarial Antigen

Table 2. Humoral antibody detection by IHA and ELISA

397

Group

IHA ELISA

Mean titre +__ S.D. (range) Mean A ~ _+ S.D. (range) P

I Antigen +TDM 8192 _+ 1644 16,384 _+ 13,653 1.403 _+ 0.102 (16,384 - 4096) (32,768 - 8192) (1.39 - 1.42)

II Antigen 1024 __+ 853 8192 __+ 7509 1.003 __. 0.113 (2048 - 5 1 2 ) (16,384 - 4096) (1.00 - 1.02)

III TDM 32 __+ 14 32 _+ 28 0.318 __. 0.162 (32 - 8) (32 - 16) (0.30 - 0.33)

IV Saline 16 _+ 11 8 + 7 0.215 ___ 0.106 (16 - 4) (8 - 4) (0.20 - 0.24)

0.02

0.05

Arithmetic mean for 7 animals per group. Antibody titres below 1 : 64 were considered negative. An A4~ was measured at the corresponding ELISA titre. An A400 of <0.4 was considered negative. Significance was calculated on the basis of Student's t-test (Group I versus Group III and Group II versus Group IV).

Haq, Siddiqui & Ahmad, 1984). The reaction intensity was observed by measuring the diameter, thickness of erythma and indurat ion at the skin test site after 12, 24, 48 and 72 h o f antigen inoculation. Skin tissues from the reaction sites were sectioned and stained with iron hematoxylin and eosin for histological studies.

Leukocyte migration inhibition test (LMIT) After performing the delayed skin test, the

leukocytes were obtained f rom rabbits by sedimentation o f heparinised blood with 6°7o dextran and further purified by centrifugation on Ficoll - Hypaque (Sigma) for 30 rain at 400 g. These polymorphonuclear leukocytes were washed 3 times with Hank ' s balanced salt solution (HBSS) and finally suspended in R P M I 1640 medium at a concentrat ion of 4 × l0 s ceUs/ml. The 75/~1 capillaries filled with cell suspension and centrifuged at 100g for 5 min, were cut at the c e l l - f l u i d interface and placed in a Mackaness migrat ion chamber containing R P M I 1640 medium supplemented with 10% foetal calf serum. The medium also contained 15/ag per ml o f antigen protein and antibiotics (streptomycin and penicillin). The cells were allowed to migrate for 48 h at 37°C and the areas of migrat ion projected, traced and quantif ied by planimetry. In control chambers the filarial antigen was omitted. The percentage of leukocyte migrat ion inhibition was calculated as follows:

% LMI=100- area of migration with antigen × 100. area of migration without antigen

Macrophage migration inhibition test (MMIT) After bleeding the rabbits for isolation o f

leukocytes, 2 rabbits o f each group were injected with 25 ml of sterile paraffin oil i.p. Four days later, peritoneal exudate cells (PECs) were obtained by injecting about 50 ml of ice cold heparinised (5 uni ts /ml) HBSS into the peritoneal cavity. The abdomen was shaken for 5 rain, then peritoneal fluid was aspirated and pooled into a separating funnel and allowed to settle for 1 h. The aqueous phase was separated and centrifuged at 1000revs /min for 10 rain at 4°C in sterile disposable plastic tubes. The centrifuged cells were pooled and washed with HBSS twice. After the last wash, PECs were suspended in RPMI 1640 media at a concentrat ion of 4 × 106 cells/ml. Capillaries were filled with cell suspension and experiments were performed similarly as leukocyte migration inhibition. The percentage o f MMI was also calculated with the same formula.

R E S U L T S

The I H A titre values in all the rabbits subjected to filarial antigen plus T D M combinat ion was higher (mean titre value 8192) as compared to Group II animals which showed a titre o f 1024 only. Similarly, the ELISA titre was 16,384 in Group I animals. The rabbits which received antigen only (Group II) showed a mean ELISA titre o f 8192. The sera of rabbits given either T D M or saline were negative (less than 64) for humoral antibodies (Table 2).

All the rabbits in Group I showed positive delayed type skin reaction. These studies showed around a

398 A. SHARMA and S. N. UPADHYAY

Table 3. CMI detection in vivo by delayed skin test

Group Mean lesion diameter Mean induration thickness

(mm ± S.D.) (mm _ S.D.)

I Antigen + TDM 13.05 ± 0.53 3.89 ± 0.76 II Antigen 5.03 ± 0.89 1.50 +_ 0.65 III TDM 3.87 _ 1.12 1.30 _ 0.88 IV Saline 2.56 _+ 1.30 1.28 ___ 0.96

Arithmetic mean of 7 experiments ± S.D. Skin reaction diameter and induration thickness of less than 5 and 2 mm, respectively, were taken as negative.

Table 4. CMI detection by LMIT and MMIT in vitro

Group

Mean leukocyte Mean macrophage migration inhibition migration inhibition

(%) _+ S.D. (%) _+ S.D.

I Antigen + TDM I1 Antigen llI TDM IV Saline

48.22 +_ 1.15 52.12 ± 1.21 14.81 ± 1.67 17.94 ± 0.94 6.44 _+ 2.04 8.09 ± 1.13 4.33 + 1.81 3.86 +_ 1.02

Arithmetic means of 7 animals per group. *P<0.01 versus Group III /°<0.05 versus Group IV.

p*

<0.01 <0.05

61°70 increase in delayed hypersensitivity in animals immunized with the a n t i g e n - T D M combinat ion as compared to rabbits which received antigen only (Table 3). A positive skin reaction of a diameter and indurat ion thickness greater than 5 and 2 mm was characterized by a zone of erythma and induration, respectively. These reactions were found to be maxi- mum at around 48 - 55 h, subsiding thereafter. Histo- logical examination of the skin reaction site showed characteristic perivascular cuffing with prominent cell infiltration, of lyrnphocytes and macrophages.

The detection of cellular immune responses were further confirmed by in vitro tests. Leukocytes and macrophage migrat ion inhibition tests showed 48.22 and 52.1207o migrat ion inhibition values in the rabbits which received an antigen plus T D M combination. The per cent increase of LMI and M M I in Group I animals, however, was calculated to be 69 and 6607% respectively, as compared to Group II animals which received antigen only. Filarial antigen, T D M , or saline alone could neither induce cellular immune responses in vivo nor in vitro (Table 4).

DISCUSSION

There are limited reports available on experimental filariasis and studies of their immune responses in animals. Most of the reported immune

response studies were carried out in human filariasis. Earlier, it has been reported that Setaria cuticle or sheath serves as an absorptive (Chen & Howells, 1979) and immunological ly active layer (Mackenzie, Proston & Ogilvie, 1978; Howells, 1980; Ogilvie et al., 1980; Parkhouse, Philipp & Ogilvie, 1981). Purified heterologous filarial antigens showed specificity with human parasites for their use in immunodiagnosis (Ottesen, 1984). These studies have included the use of S. digitata antigens (Dissanayake & Ismail, 1980). Several scientific workers have used S. digitata as a filarial antigen to raise antibodies and for immunodiagnosis of bancroft ian filariasis (John Bright & Kaleysaraj, 1990; Gerald Theodore & Kaliraj, 1990). The present investigation is a maiden at tempt of using an aqueous suspension of T D M as a very good immune stimulant against filariasis. Our results showed that S. digitata sheath antigen alone can only induce humoral response in rabbits. But the combinat ion of antigen with an immunoadjuvant TDM, elicited both humoral and cellular responses.

Earlier studies have shown specific cellular immune unresponsiveness in human W. bancrofti infection (Ottesen, Weller & Heck, 1977). Later Piessens et al. (1980) reported a correlation between cellular and humoral reactions to microfilarial antigen with clinical status. The same workers (Piessens et al., 1982) reported that individuals

Immune Response

suffer ing f rom filariasis have a marked ly suppressed cel l-mediated i m m u n e response to nonspecif ic mi togens and to filarial ant igens.

Our results of cellular immun i ty against S. digitata sheath ant igen in vivo by a skin test, in vitro by L M I T and M M I T , are in good agreement with each other . This repor t fu r ther conf i rms the weak immunogen ic i ty o f a filarial ant igen alone. T D M played a m a j o r role in represent ing cellular response at a s ignif icant level. In our opin ion , the h igh ti tre ant i f i lar ia l sera could be used as a tool for detect ing

and a Filarial Antigen 399

the ES ant igen in filarial pat ients . Since, in our exper iments bo th the i m m u n e responses are signi- f icantly positive, these f indings could be of great help in pro tec t ion studies in future . However , we do not have any da ta to suppor t it.

Acknowledgements - - The authors are grateful to Dr Aditi Sarkar, School of Biochemical Engineering, I.T., B.H.U., Varanasi for helping in animal immunizations. This work was financially supported by University Grants Commission, New Delhi, India.

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