Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs

Research in Veterinary Science /990, 49, 92-97

Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs D. IVANOSKA, K. CUPERLOVIC, Institute of Endocrinology, Immunology and Nutrition, INEP, Banatska Llb, 11080 Zemun, Yugoslavia, J. K. LUNNEY, USDA, Agricultural Research Service, LPSI, Helminthic Disease Laboratory, Beltsville, Maryland 20705, USA

The immune response of 'Yugoslav meat breed' pigs inoculated with low doses of Trichinella spiralis muscle larvae was followed over two to nine weeks of primary infection, by analysing changes in peripheral blood mononuclear cell subsets, the development of a humoral antibody response and muscle larvae burden. During the course of the infection, infected animals showed a persistent elevation of both CD4 + and CDS + T cell subsets from days 15 to 60 after the parasite exposure. During this time, the number of peripheral blood mononuclear cells expressing major histocompatibility complex class II antigens was also increased, while no significant differences were found in the number of circulating monocytes/macrophages and B cells over time. Humoral antibody responses to muscle larvae excretory-secretory products were evident as early as 41 days after infection, while the muscle larvae were recovered as early as 27 days after infection. The increased levels of CD4 + and CDS + T cell subsets, as well as cells expressing major histocompatibility complex class II antigens in pigs exposed to T spiralis, may be indicative of some considerable alterations in cell subsets that are involved in the regulation of the swine immune response to this parasite. THE immune response to helminthic parasite infection is complex, involving immune effector mechanisms ranging from activation of specific T cell populations and of antibody production, to nonspecific production of inflammatory mediators by mast cells and eosinophils, and thus can be analysed in several different ways. One approach to understanding the complex events following infection is the analysis of changes in cell subsets involved in immune reactions, since many parasitic infections are associated with notable alterations in the distribution of lymphocyte subpopulations (Cohen 1982). Many studies, facilitated in the last few years by development of monoclonal antibodies (mAb) that recognise particular cell populations, verify that changes in peripheral blood cell subsets may provide relevant 92

clinical and immunological information about host responses to infectious diseases (Piessens et al 1982, Ohta et al 1983, Lunney et a11986, Ellis et aI1987). The availability of mAb specific for porcine lymphoid cell subpopulations (Lunney and Pescovitz 1988) allowed the present authors to analyse sequential changes in different peripheral blood cell subsets following Trichinella spiralis infection in this species. In addition, mAb that recognise swine major histocompatibility complex (MHC or SLA) antigens (Lunney et al 1988) can be useful reagents for detecting activated cell populations and therefore for gauging the response to a parasite. Swine infected with T spiralis generally conform to the classical pattern of infection dynamics described for other host species, but they also show some potentially important differences in the immune response to this parasite. Namely, compared with rodents, pigs exhibit a delayed and a lower antifecundity response and intestinal expulsion of adult worms, especially at low dose infection (Murrell 1985, Marti and Murrell 1986) and the anti-newborn larval response is the major component of their protective immune response (Marti et al 1987). It was suggested that the persistence of intestinal worms for four to six weeks after infection (Murrell 1985) could be due to a lower level of gut immunity resulting in much heavier muscle larvae burden of T spiralis in this species. Determination of immunoregulatory mechanisms involved in disease response and enhanced resistance to this parasite is therefore of greater importance in swine. This paper presents an analysis of peripheral blood mononuclear cell (PBMC) subpopulations during the course of Tspiralis infection in 'Yugoslav meat breed' pigs. The animals were also analysed for the development of a humoral antibody response to muscle larvae excretory-secretory products, and parasitologically examined for muscle larvae recovered from their diaphragms to confirm the infection, since in pigs, unlike humans and experimental rodents, the trichinella infection is associated with no clinical symptoms regardless of the intensity of infection.

Cell subsets in T spiralis-injected pigs

93

TABLE 1: Monoclonal antibodies used to analyse porcine peripheral blood mononuclear cell subpopulations mAb

Ig class

Specificity

Reference

74-12-4 76·2·11 74-22-15 400 TH16 5C9 Polyclonal anti-pig Ig

IgG2b, k IgG2a, k IgG2b, k IgG2a, k IgG2a, k IgG 1, k

Porcine C04 + T cells Porcine C08 + T cells Macrophages, granulocytes SLA-DR, class II MHC SLA-DQ, class II MHC B cells, pig IgM + cells B cells. pig Ig + cells

Pescovitz et all1984, 19851 Pescovitz et all1984, 19851 Lunney and Pescovitz 119881 Lunney et at 119881 Lunney et a111988) Paul et at 119851

mAb MHC

Monoclonal antibodies Major histocompatibility complex antigens

Materials and methods

Fluorescentanalys~

Animals and experimental protocol

The specificities of mAb used to detect surface antigens on porcine PBMC are listed in Table I. For indirect immunofluorescent staining, cells (1 x 1()6) were centrifuged and the pellet resuspended in 25 J-li of individual mAb or in medium, as a control. After a 45 minute incubation at 4°C, the cells were washed twice in the staining medium (PBS with 0·1 per cent bovine serum albumin and 0'1 per cent sodium azide) and then mixed with 25 J-ll fluorescein (rrrcj-conjugated sheep anti-mouse IgG (lNEP). Fluorescein (FITC)conjugated sheep anti-swine IgG (INEP) was used to directly label surface immunoglobulin-positive B cells. After a 30 minute incubation at 4°C, the cells were washed twice, resuspended and analysed for surface fluorescence under an ultraviolet microscope. A total of at least 200 cells was counted for each sample. The percentage of positive cells was calculated by subtracting the background, mediumonly stained cells, which, normally, were less than 3 per cent.

Domestic 'Yugoslav meat breed' pigs were raised at Topola, Backa Topola. The pigs were of either sex, matcher for age and weight, and were treated to be helminth-free. T spiralis (a strain isolated from infected swine muscles at a Belgrade slaughterhouse in 1976 and kept by periodical passage in Wistar rats) infective muscle larvae (Ml) were prepared from car cases of infected rats and inoculated by oesophageal intubation. Twelve pigs received Ml recovered by hydrochloric acid-pepsin digestion from infected rat muscle, while four uninfer.ted control animals were maintained at the same lot throughout the experiment. Each animal was weighed and bled at the start of the experiment (day 0), and then on days 15,21,41 and 60 thereafter, for the analysis of PIIMC subpopulations and serological testing. Starting from day 27, the pigs were gradually killed and parasitologically examined by enzymatic digestion of their diaphragms for Ml recovery. AIK

Competitive inhibition assay (CIA) Peripheral blood sampling Blood samples were collected from a jugular vein. PBMC were separated from whole heparinised blood by density gradient centrifugation in a FicollRonpacon. As the use of cryopreserved cells was a necessity under the experimental conditions of this study, each test day the PIIMC from each animal were frozen in DMEM medium containing 20 per cent fetal calf serum, 25 mM HEPES and 10 per cent dimethylsulphoxide, by direct placement at - 80°C (Ivanoska et al 1987). Before use (approximately 70 days after freezing for each sample), frozen cells were thawed rapidly at 37°C, washed in phosphate-buffered saline (PIIS) with I per cent fetal calf serum, and then analysed for fluorescent staining. Cells retained good viability and surface marker expression after thawing, as previously described (Ivanoska et al 1987).

Serum samples obtained from each pig were analysed for antibody reactivity to T spirafis Ml excretory-secretory (ES) antigens by CIA (Cuperlovic et al 1987). Briefly, plastic microtitre plates were coated with ES antigens obtained after the in vitro cultivation of T spiralis Ml. Antibody responses to Ml ES antigens were analysed by incubating pig sera and 1251-labelled anti- T spirafis ES mAb, 7C 2C5 (Gamble and Graham 1984) in the antigen-coated wells for five hours. The percentage inhibition of 7C 2C 5 mAb binding was calculated as follows:

010 inhibition = 100 cpm bound with test serum wi h neaati negative control sera x 100 mean cpm b oun d Wit Percentage inhibition greater than the mean (2,91 per cent) + 7 SD (6'12) of 20 serum samples from T

94

D. Ivanoska, K. Cuperlovic, J. K. Lunney

TABLE 2: Trichinella spiralls muscle larvae IMLllnoculatlon dose. larval recovery and temporal appearance of antibodies In Yugoslav meat breed pigs Weight (kg)

Group (n)

31·S" ± 1· 3 37'8 ± 0'3 31'5 ± 1·4

Control (41 Lower dose (21 Higher dose (101

Antibody detection (day)

Inoculation ML kg- 1

Total ML

100 2·7 0·0 0·0 ± 11 ·1 340 ± 1 ·8 ± 50 ±

Recovery Ipg

0

15

21

41

SO

0·0 0·0 28'5 ± 14·4

0/4t (0) 0/2 (0) 0/10 (4'SI

0/4 (5 ·1) 0/2 (13' 7) 0/10 (4'2)

0/4 (2·3) 0/2 (S·91 0/10 (3·2)

0/4 (1'3) 0/2 (5'S) 5/7 (52'3)

0/4 (8'51 011 (0) 515 (91·8)

±

Ipg Larvae g - 1 diaphragm tissue " Data are presented as group mean ± SEM t Data are presented as number positivelnumber of total tested sera (mean percentage of inhibition in CIA)

spiralis-negative pigs (45' 75 per cent) was established as the value for positive reactivity. Results

were recovered when they were killed. For the remaining 10 pigs the infection was confirmed by antibody detection (seropositive as early as 41 days after infection) and, or, parasitological findings (ML recovered as early as 27 days after infection).

Parasitological and serological parameters of infection

Analysis of PBMC subsets

Twelve 'Yugoslav meat breed' pigs were inoculated with Tspiralis ML (100 to 500 total). The animals were analysed for their antibody response on days 15,21, 41 and 60, and parasitologicaIly examined for ML recovered from the diaphragm starting from day 27 after infection. For two pigs which received the lowest inoculation dose (Table 2), evidence of infection could not be found, as they failed to be seropositive and no ML

Sequential changes in PBMC subsets were analysed during the course of this porcine T spiralis infection. The analysis of PBMC with mAb defining T lymphocyte subsets revealed that, following infection, there was a significant increase in the number of both CD4 + and CDS + T cell subsets. As shown in Fig la, there was a continuous elevation of CD4 + T cells in all animals tested. However, whereas the control pigs exhibited a statistically significant increase of CD4 +

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FIG 1: Sequential analysis of T cell subsets following Trichinella spiralis inoculation: (a) CD4 + T cell subset; (b) CD8 + T cell subset; (c) CD4/CD8 T cell ratio. Graphs indicate percentage of positive cells in peripheral blood; values are the mean ± SEM. (0) Controls. (.) lower dose and ( • I higher dose inoculation

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cells on day 60 of the experiment compared to day 0 values, infected animals had significant elevations of CD4 + cells starting as early as day 21 after infection. As predicted from the lack of parasitic recovery, two animals with a lower dose ML inoculation exhibited a similar lower elevation of CD4 + T cells. Similarly, the percentage of CD8 + T cells showed a permanent increase, but only in the infected animals (Fig Ib). Compared to pre-infection levels, significant increase of PBMC CD8 + T cells in the higher-dose infected animals was observed on days 41 and 60, whereas only a slight increase was noticed in the lower dose animals and the control. The assessment of the CD4!CD8 T cell subset ratio confirmed that these pigs, like other normal pigs, exhibited a low ratio (0'69 ± 0'04) on day O. Although in all animals it increased with age to O' 87 ± 0·05 on day 21, it has never reached the level of I· 5 exhibited by normal humans. In animals exposed to T spiralis ML inoculum, there was the initial age-induced increase which remained relatively constant after 21 days of infection, reflecting the concomitant increase of CD4 + and CD8 + T cells in the PBMC (Fig l c). The use of mAb reactive with antigen expressed on porcine monocytes!macrophages and granulocytes, produced no significant differences over time in the numbers of circulating PBMC monocytes!macrophages of either group (data not shown). The analysis of PBMC with B lymphocyte-specific reagents (anti-

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FIG 3: Sequential analysis of cells expressing class II major histocompatibility complex antigens following Trichinella spirelis inoculation: (a) cells positive with anti·SLA-OR mAb; (b) cells positive with anti-SLA-OQ mAb. Graphs indicate percentage of positive cells in peripheral blood; values are the mean SEM. 0 Controls•• lower dose and • higher dose inoculation

=

pig IgM mAb, ann-pig Ig polyclonal antiserum) revealed some variations in the percentage of positive cells, but there were no overall changes in either group (Fig 2). However, it was noticed that all animals had higher apparent B cell levels when defined by polyclonal antisera, and that infected animals exhibited a slight elevation of B cell content in comparison to the control. . Changes in the numbers of PBMC that express MHC or SLA class II products were examined by analysing PBMC reactivity with two mAb (40D, anti-st.A.na, and TH 16, ann-stx-oo) which recognise two independent sets of antigens. Slight differences were observed in the reactivity pattern of cells staining for these two class II specific mAb. Levels of class II positive cells defined with anti-sLA-DR specific mAb (Fig 3a) were increased only in 10 T spiralis-infected pigs, although no statistically significant differences were detected. A similar increase in the percentage of class II positive cells defined by the anu-si.x.oo mAb was observed in all tested animals, regardless of the treatment applied (Fig 3b).

Discussion The analysis of the PBMC subpopulations following

T spiralis primary infection demonstrated that some potentially important changes in cellular subsets have

96

D. Ivanoska, K. Cuperlovic, J. K. Lunney

been evoked during the immune response of experimentally infected 'Yugoslav meat breed' pigs. Infected animals exhibited the most consistent and marked alterations in peripheral T cell subsets. Namely, from days 15 to 60 after infection, these animals showed a persistent elevation of both CD4 + and CD8 + T cell subsets. As the animals used were only two to four months old during the experiment, an age-dependent increase of CD4 + T cells occurred even in uninfected pigs, replacing some of the null T cell content (lvanoska et al 1987). However, it should be noted that such an increase was much lower and delayed in relation to the elevation seen in the infected groups. The differences in the dynamics and mutual levels of CD4 + and CD8 + T cell subsets observed in the infected and the control animals were also characterised by differences in the CD4/CD8 ratio found in these groups. Due to a proportionally greater increase of CD4 + T cells an increased ratio was exhibited by the controls, whereas an almost simultaneous elevation of both T cell subsets in the infected pigs accounted for a less elevated level of CD4/CD8 ratios. Although variable over time, the means of all groups always remained less than one. Such a low level of CD4 + to CD8 + T cell ratio is one of the unique characteristics which have normally been described for resting swine T cells (Pescovitz et al 1985, Saalmiiller et al 1987). The functional implication of the inverse ratio, as compared to humans, is unknown at present. The concomitant elevation of both CD4 + and CD8 + T cells detected in T spiralisinfected pigs could be associated with the rise of both cell subsets or a CD4 + CD8 + subset. Another unique characteristic of swine T cells is that an unusually high number (8 to 64 per cent) of CD4 + CD8 + dual expressor T cells was found in the resting peripheral T lymphocytes of normal pigs (Pescovitz et al 1985, Saalmiiller et al 1987). Whether this parasitic infection influences the accumulation of the dual positive CD4 + CD8 + population remains to be determined. The role and genealogy of these porcine dual expressor T cells in the immune function is also unknown. Some data indicated that these cells were unlikely to be precursors for CD4 + CD8 - and CD4 - CD8 + cells and that they did not make a major contribution to the cytotoxic T lymphocyte activity (Pescovitz et al 1985); it has also been shown that the expression of the CD8 antigen corresponded to that of the CD4 - CD8 + dull population, eg, population of CD8 + T cells that express approximately a quarter the amount of antigen (Pescovitz et al 1985, Saalmiiller et aI1987). In humans and rats T lymphocytes with that phenotype were identified as blast-like cells, linked to activation (Blue et al 1985, 1986, Bevan and Chisholm 1986), and for humans it was further suggested that the CD8 + dull population had a suppressor function (Lunney and Pescovitz

1988). Although the functional significance of these differences (both the high percentage of CD4 + CD8 + population and the excess of CD8 + T cells) in normal pigs is unknown at this time, the increase of CD8 expression on porcine PBMC following T spiralis infection could possibly reflect the elevated level of cells expressing suppressive phenotype. In the course of the infection, T spira/is-infected pigs exhibited no overall changes in the numbers of circulating monocytes/macrophages and B cells. However, some increase was noticed in the level of B cells determined with polyclonal anti-swine Ig reagent. Since there was no corresponding elevation of the number of B cells defined with anti-pig IgM mAb, this result is likely to suggest that cell surface Fc receptors for immunoglobulins may have been increased, or that serum Ig levels have increased, so that normally empty Fe receptors now bind pig Ig. The elevation of levels of the PBMC reactive with the two SLA class II specific mAb was also demonstrated in Tspira/is-infected pigs. This is in agreement with some former experiments which suggested that increased levels of class II expression, and not necessarily increased numbers of class II positive PBMC, might be an important predictor of activated cell populations for this helminthic infection, too (J. K. Lunney and K. D. Murrell, unpublished data). Because of the difficulty of visually assessing the intensity of class II staining, the percentage of PBMC bearing class II could not be accurately quantitated. As no overall increase of the number of monocytesl macrophages and B cells was observed in the present experiment, the elevated level of class II positive PBMC probably largely reflected the T cell dynamics. Resting swine T cells express class II antigens constitutively, and it has been shown that more than 90 per cent of CD8 + T cells express SLA-DR and about 70 per cent SLA·DQ antigens, while about 40 per cent of CD4 + T cells are positive for SLA·DR and 30 per cent for SLA-DQ antigens (Lunney and Pescovitz 1988). Further demonstration of the expression of class II molecules on the separated T cell subsets during the course of the infection has to be made, as the distribution of these antigens on porcine T cells tends to differ from other species and their functional significance is unknown. The consistent elevation in levels of both T cell subsets and class II positive PBMC in the T spiralisinfected pigs may indicate some considerable alterations of cell subsets involved in the antigen and disease response of this species, as the unique composition of the porcine T lymphocyte population has to be taken into consideration when porcine immune effector mechanisms are analysed. In pigs infected with Tspiralis evidence of differential immune responsiveness (such as a delayed and

Cell subsets in T spiralis-injected pigs lower intestinal expulsion of adult worms and lack of clinical symptoms) indicate that the balance of the host-parasite system is potentially established at a different level when compared with other host species (humans, experimental rodents). In pigs, parasite survival and proliferation seem to be favoured, but what the mechanisms of immune evasion are is hard to say. The differences of immune response observed in infected pigs could be related to poor stimulation of inflammatory responses against parasites, components of which seem to cause expulsion of worms (Wakelin 1978, 1985), as well as to hypersensitive reactions which influence the onset of clinical signs. Though it appears controversial, these reactions may have a protective role in parasitic infections. Immunosuppression is one of the possible mechanisms of immune. evasion and also an important aspect of immunopathology. In pigs, there is little evidence for T suppressor cells and their function, but the understanding of their role is, no doubt, of scientific interest, Acknowledgements We thank Dr H. R. Gamble for providing the 7C 2CS monoclonal antibody. This work was supported in part by a grant from USDA, OICD, SFCRP (JFP-713 and FG-yu-265), and by RZN SR Serbia. References' BEVAN, D. J. & CHISHOLM, P. M. (1986) Immul/ology 59, 621-625 BLUE, M. L., DALEY, J. F., LEVINE, H. & SCHLOSSMAN, S. F. t1985) Journal of Immunology 134, 2281-2286 BLUE, M. L., DALEY, J. F., LEVINE, H., CRAIG, K. A. & SCHLOSSMAN, S. F. (1986) Journal of Immunology 137, 1202-1207

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COHEN, S. (1982) Immunology of Parasitic Infections. Oxford. Blackwell Scientific. pp 138-161 CUPERLOVIC, K., MOVSESIJAN, M., IVANOSKA, D., SOFRONIC, L. & JOVANOVIC, B. (1987) Veterinarski arhiv 57, 133-141 ELLIS, J. A., SCOTT, J. R.,MacHUGH, N. D.,GETTINBY, G.& DAVIS, W. C. (1987) Parasite Immunology 9,363-378 GAMBLE, H. R. & GRAHAM, C. E. (1984) American Journal of Veterinary Research 45, 67-74 IVANOSKA, D.,CUPERLOVIC, K.& LUNNEY, J. K. (1987)Ac/a Veterinaria (Beograd) 37,263-274 LUNNEY, J. K. & PESCOVITZ, M. D. (1988) Differentiation Antigens in Lymphohemopoietic Tissues. New York and Basel, Marcel Dekker. pp 421-454 LUNNEY, J. K., SUN, D. c., IVANOSKA, D., PESCOVITZ, M. D. & DAVIS, W. C. (1988) The Molecular Biology of the Major Histocompatibility Complex of Domestic Animal Species. Ames, Iowa, ISU Press. pp 97-119 LUNNEY, J. K., URBAN, J. F. & JOHNSON, L. A. (1986) Veterinary Parasitology 20, 117-131 MARTI, H. P. & MURRELL, K. D. (1986) Experimental Parasitology 62,370-375 MARTI, H. P., MURRELL, K. D. & GAMBLE. H. R. (1987) Experimental Parasitology 63,68-73 MURRELL. K. D. (1985) Experimental Parasitology 59,347-354 OHTA. N., MINAI, M. & SASAZUKI, T. (1983) Journal of Immunology 131, 2524-2528 PAUL, P. S., VAN DEUSEN, R. A. &MENGELlNG, W. L. (1985) Veterinary Immunology and Immunopathology 8, 311-328 PESCOVITZ, M. D., LUNNEY, J. K. & SACHS, D. H. (1984) Journal of Immunology 133, 368-375 PESCOVITZ, M. D., LUNNEY, J. K. & SACHS, D. H. (1985) Journal of Immunology 134, 37-44 PIESSENS. W. F., PARTONO. F., HOFFMAN, S., RATIWAYANTO. S., PIESSENS, P. W., PALMIERI, J. R.. KOlMAN. I., DENNIS, D. T. & CARNEY. W. P. (1982) New England Journal of Medicine 307, 144-148 SAALMULLER, A., REDDEHASE, M. J .• BUHRING. H. J .• JONJIC, S. & KOSZINOWSKI, U. H. (1987) European Journal of Immunology 17, 1297-1301 \V AKELlN, D. (1978) Nature 273,617-620 WAKELlN, D. (1985) Parasitology Today I, 17-23 Received September 5, 1989 Accepted December 4, 1989