Journal of Neuroimmunology, 37 (1992) 93-97 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

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JNI 02140

Evidence of altered T-lymphocyte number and proliferative responses in genetically epilepsy-prone rats Seddigheh Razani-Boroujerdi ~, Raymond R.R. Rowland ~'~, Karen A. Ortiz h, Daniel D. Savage h and Sei T o k u d a ~ Departments of a Microbiology and h Pharmacology, Unirersity of New Mexico School of Medicine, Albuquerque, NM 87131, USA (Received 22 July 1991) (Revised, received 15 October 1991) (Accepted 15 October 1991)

Key words." T Lymphocyte; Concanavalin A; Pokeweed mitogen; Epilepsy

Summary Genetically epilepsy-prone (GEPR-9) rats exhibit decreased antibody plaque-forming cell responses following immunization. We examined the hypothesis that this immunosuppression was due to deficits in the number or proliferative responses of T-lymphocytes. Splenocyte responses to concanavalin A and pokeweed mitogen were significantly greater in GEPR-9 rats than controls. Flow cytometric analysis indicated that GEPR-9 rats possess an increase in T-cells associated with the T-helper phenotype. The increased proportion of T-helper cells in GEPR-9 rats may underlie their enhanced proliferative responses to T-cell mitogens. These results clearly indicate that the failure of the GEPR-9 rat to respond to a T-dependent antigen in vivo is not due to a lack of T-helper activity.

Introduction The genetically epilepsy-prone (GEPR-9) rat is a genetic model of epilepsy derived from selective inbreeding of Sprague-Dawley rats for audiogenic seizure susceptibility (Jobe et al., 1973; Reigel et al., 1986). Both noradrenergic (Jobe et al., 1973, 1982) and thyroid hormone deficits (Mills and Savage, 1988) have been implicated as etiological

Correspondence to: Sei Tokuda, Ph.D., Department of Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. i Current address: Department of Microbiology, University of Minnesota, Minneapolis, MN 55455, USA.

factors contributing to the development of the seizure-prone state in GEPR-9 rats. The nature and degree of these neuroendocrine abnormalities coupled with unpublished observations of recurring infections and reduced spleen size in GEPR-9 rats prompted an investigation of their immune system. Preliminary experiments revealed diminished direct and indirect antibody plaque-forming cell responses following immunization with sheep erythrocytes (SE) in GEPR-9 rats as well as decreased levels of circulating IgM in unimmunized GEPR-9 rats compared to control (Rowland et al., 1991). A decreased response to a T-lymphocyte-dependent antigen such as SE raised the possibility that GEPR-9 rat T-cells may not func-

not function normally. Howevcr, our previous study revealed that unimmunized G E P R - 9 rats possessed increased lcvels of circulating lgG. This paradoxical observation presented the alternative possibility that T-cell activity might be enhanced in G E P R - 9 rats. We examined these questions by evaluating the proliferative response of spleen cell cultures stimulated with concanavalin A (ConA) or pokewccd mitogen (PWM). ConA is a T-cell mitogen which evaluates the ability of T-cells to respond to antigen. PWM is a T-cell-depcndent B-cell mitogen which is used to study the interaction between T-helper cells and B-cells. In addition to measuring lymphocytc proliferative rcsponses, the proportions of T-cells expressing thc T-helper and T-suppressor phenotypes were measured by flow cytomctric analysis.

Materials and methods

Animals Non-cpileptic Sprague-Dawley control and G E P R - 9 rats were derived from breeding colonics maintained in the Animal Care Facility of the University of Ncw Mexico School of Medicinc. Rats wcrc maintaincd on a 12 h light (07.00-19.00 h), 12 h dark cycle and given water and Purina breeder block chow ad libitum. Control and G E P R - 9 rat progeny wcrc screened for audiogenic seizure susceptibility at 45 days of age according to the method of Jobe et ai. (1973). The non-epileptic control rats exhibited an audiogenic response score (ARS) of zero (no seizure in response to an acoustic stimulus). G E P R - 9 rats exhibited an ARS score of 9 (full tonic extensor motor seizure in response to an acoustic stimulus). After testing, the rats were allowed to recover for a minimum of 6 weeks prior to the immunological studies.

Cell preparation Spleen and mesenteric lymph nodes were removed from l(10-130-day-old control and G E P R 9 rats and immediately put in ice-cold RPMI 1(-,40. The tissues were minced and pressed through a nylon sieve (pore size 110 /.zm). The resulting suspension was frccd of cellular aggre-

gatcs and debris by passage through a nylon filter (pore size 40 p.m). Red blood cells were removed by lysis in 0.83¢4 ammonium chloride for 2 rain. The cell suspension was washed 3 times and resuspended in complete tissue culture medium. The complete tissue culture medium for lymphocyte proliferation contained RPMI 16411 supplemcnted with 111eA heat-inactivated fetal bovine serum. IN mM Hepcs, 15 mM sodium bicarbonate, 5(I units/ml penicillin, 50 # g / m l streptomycin, 2 mM ~.-glutaminc, 1 mM sodium pyruvatc and 0.1 mM non-essential amino acids.

Mitogen-induced proliferation assays Proliferation assays were carricd out in 96-wcll fiat-bottom microtitcr culture plates (Costar, Cambridge, MA, USA). All experiments wcrc performed in triplicate. 100 #1 of sp[enocytc suspension containing IW' cells were added to wells with 10(I /xl of medium containing various concentrations of ConA (Sigma Chemical Company, St. Louis, MO, USA) or PWM extract (Gibco Laboratories, Grand Island, NY, USA). The plates were incubated at 37°C for 72 h in a humidified 5% CO~ and air atmosphere. 200 nCi of [~H]thymidine (specific activity 20 C i / m m o l , Dupont-NEN, Boston, MA, USA) were added to each well 24 h prior to harvesting with a semiautomatic multiple sample harvester (Skatron Instruments, Sterling, VA, USA). [~H]Thymidine incorporation was measured in a Beckman liquid scintillation counter. The group means were compared statistically by Student's paired t-test using the A B S T A T statistical amdysis program (Anderson-Bell. Parker, CO, USA).

How cytometric analysis 200/~1 suspensions from spleen or lymph node containing 1 × 107 cells were incubated for 30 min at 4°C with 511 /zl of a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Serotcc-Bioproducts, Indianapolis, IN, USA). The antibodies used were: (1) O X I 9 to label total T-cells (CD5 ÷ cells), (2) W 3 / 2 5 to label T - h e l p e r / i n d u c e r cells (CD4 ~ cells). (3) M R C OX8 to label T - s u p p r e s s o r / cytotoxie cclls ( C D 8 - cells). Cells were washed twice with Hanks' balanced salt solution and fixed with 1% paraformaldchydc in saline. Fluorcs-

95

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cence cell analysis was performed using Becton-Dickinson FACScan flow cytometer.

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Mitogen-mduced proliferation responses The threshold and peak concentrations for the proliferative response of spleen cells to ConA were similar between the two groups. However, the magnitude of the response was increascd significantly in GEPR-9 rats compared to control rats (Fig. ! A). At 5 . 0 / z g / m l , the maximally stimulating concentration of ConA, lymphocyte proliferation in GEPR-9 rats was nearly 2-fold greater than control. The peak proliferative response to PWM ex-

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tract was also nearly 2-fold greater in GEPR-9 rats (Fig. 1B). However, the peak response in GEPR-9 rats occurred at 2.5 /zg/ml as opposed to a peak response at 1 0 / z g / m l in control rats.

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POKEWEED MITOGEN (~L/mL) Fig. 1. Mitogen-induced splenic lymphocyte proliferation at different concentrations of ConA ( A ) and PWM extract (B) in non-epileptic Sprague-Dawley control (open squares) and GEPR-9 rats (filled squares). Each data point represents the mean_+ the standard error of the mean of six rats from each group. Asterisks denote data significantly different than control based on a Student's paired t-test (* p < 0.01).

FACScan flow cytometric analysis revealed a significant increase in the number of OX19 ÷ (total T) ceils in G E P R - 9 splenocytes compared to control (Fig. 2A). The relative number of W 3 / 2 5 ÷ (T-helper) ceils was also increased, whereas the percent of OX8 ÷ (T-cytotoxic and T-suppressor) ceils was not different. An elevation in the proportion of W 3 / 2 5 + ceils was also observed in the lymph nodes (Fig. 2B).

Discussion Our previous studies (Rowland et al., 1991) demonstrating immune suppression in the

9fi

G E P R - 9 rat suggested a possible defect in Thelper cells or their ability to communicate with B-cells. However, the results in Fig. 1A indicate that the G E P R - 9 splenocytc proliferative response to ConA is intact. Since ConA stimulates the proliferation of all T-cells, additional experiments were performed using PWM. PWM stimulation predominantly reflects T-cell-dependent B-cell proliferation, or the ability of T-helper cells to communicate with and stimulate B-cell proliferation, differentiation and immunoglobulin synthesis (Gougen and Theze, 1986). As was observed with ConA, G E P R - 9 rat splenocytes exhibited a dramatic response to PWM (Fig. 1B). These results demonstrate clearly that the failure of the G E P R - 9 rat to respond to a T-cell-depcndcnt antigen in vivo is not due to a lack of T-helper activity. In addition, the G E P R - 9 rat splcnocytes responded to PWM at doses lower than control rats. The maximum proliferative response occurred at 2 . 5 / s g / m l in G E P R - 9 rats compared to 10 / x g / m l in control rats (Fig. I B), suggesting that G E P R - 9 rat splenocytes may have a lower threshold of responsiveness than control rats. Furthermore, the observation of increased levels of circulating lgG in unimmunized G E P R - 9 rats (Rowland et al., 1991), an immunoglobulin generally dependent on T-helper cells, is consistent with the presence of increased T-helper activity in vitro. To further examine the source of the cells responsible for the increased mitogen activity in G E P R - 9 rats, flow cytometry was used to analyze T-cell subpopulations. The rat T-cell subpopulation associated with helper function bears the W 3 / 2 5 determinant, whereas the T-cell subset associated with suppressor activity bears the OX8 determinant (Rosenberg et al., 1982). Phenotypic analysis of splenocyte populations using flow cytometry revealed that G E P R - 9 rat spleens possessed a larger percentage of W 3 / 2 5 ~ cells compared to controls whereas the percentage of OX8 + was unchanged (Fig. 2A). In both spleen and lymph node preparations (Fig. 2B) the increased number of W 3 / 2 5 + cells in G E P R - 9 rats is reflected in a corresponding increase in total T-cells. The total percentage of OX19 ÷ cells observed in the lymph node exceeded the sum of

the percentages of W 3 / 2 5 - and O X 8 ' populations (Fig. 2B). Currently, we have no explanation for this observation. However, since the T-cell populations represent a normal distribution of cells bearing variable densities of a particular marker, this discrepancy may be duc to the method used to distinguish positive and negative cells during flow cytometric analysis. In any event, the flow cytometry results suggest that there is a relative increase in the number of CD4 + T-helper cells in G E P R - 9 rats. Furthermore, the increase in ConA-induced proliferation in G E P R - 9 cultures (Fig. 1A) may be due to an increase in the number of T-cells compared to control. Mitogens arc commonly used to assess lymphocyte function. However, during a normal immune response there is the additional requirement for antigen-presenting cells (APCs), such as macrophages, and antigen to stimulate T-cell proliferation. The ability of antigen and APCs to stimulate T-cell proliferation was not examined in this series of experiments. The possibility exists that the immune defect in G E P R - 9 rats could be due to the inability of APCs to process a n d / o r present antigen to T-helper cells. In preliminary studies, we have observed no significant differences between control and G E P R - 9 rat macrophage profiles based on flow cytometric analysis of OX41 monoclonal antibody labelling of splenocytes. Subsequent experiments will evaluate the ability of G E P R - 9 APCs to process and present antigen to T-cells, as well as the response of G E P R - 9 lymphocytcs to APCs and antigen. Another possibility is that prior activation of a proportionately greater number of existing T-cells may limit the participation of T-helper cells in a response to a new antigenic challenge. Whether or not the systems that modulate T-helper activity are functionally intact in G E P R - 9 rats remains to be investigated. The involvement of the neuroendocrine system in the modulation of immunity suggests that neurological and endocrine deficits in G E P R - 9 rats could produce subsequent alterations in T cell function. Previous reports have demonstrated that G E P R - 9 rats have decreased brain monoaminc levels (Jobe et al., 1973, 1986), decreased numbers of noradrenergic receptor binding sites (Razani-Boroujerdi et al., 1988, 1989), thyroid

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hormone deficiency (Mills et al., 1988) and growth hormone deficiency (Mills et al., 1987). These factors, singly or in combination, could influence T-cells by: (1) altering the production of T-helper cells in the thymus, (2) increasing the number of T-helper cells in the periphery, or (3) changing the threshold for activation of circulating T-helper cells. Future experiments will examine these possibilities.

Acknowledgements The authors wish to thank Linda L. Paxton for technical and editorial assistance in the production of the manuscript. Supported by NS 24008, RR 08139, RR 05833 and Dedicated Health Research Funds of the University of New Mexico School of Medicine (C-1009 and C-1011).

References Gougen, T.L. and Theze, J. (1986) A polyclonal assay for T helper function involving T-B cell contact mediated by L3T4 molecules. J. lmmunol. 137, 755-759. Jobe, P.C., Picchioni, A.L. and Chin, L. (1973) Role of brain

norepinephrine in audiogenic seizure in the rat. J. Pharmacol. Exp. Ther. 184, 1-10. Jobe, P.C., Laird, H.E., Ko, K.H., Ray, T. and Dailey, J.W. (1982) Abnormalities in monoamine levels in the central nervous system of the genetically epilepsy-prone rat. Epilepsia 23, 359-366. Mills, S.A. and Savage, D.D. (1988) Evidence of hypothyroidism in the genetically epilepsy-prone rat. Epilepsy Res. 2, 102-110. Mills, S.A., Reigel, C.E., Jobe, P.C. and Savage, D.D. (1987) Deficits in serum growth hormone and postnatal growth in the genetically epilepsy-prone rats. Soc. Neurosci. Abstr., 13, 943. Razani-Boroujerdi, S., Tso-Olivas, D.Y. and Savage, D.D. (1988) Decrease in cerebellar iodocyanopindolol binding sites in 31-day-old genetically epilepsy-prone rat. Soc. Neurosci. Abstr. 18, 254. Razani-Boroujerdi, S., Tso-Olivas, D.Y., Hoffman, T.J., Weiss, G.K. and Savage, D.D. (1989) Decrease in locus coeruleus 3H-idazoxan binding sites in genetically epilepsy-prone rat. Soc. Neurosci. Abstr. 19, 46. Reigel, C.E., Dailey, J.W. and Jobe, P.C. (1986) The genetically epilepsy-prone rat: an overview of seizure-prone characteristics and responsiveness to anti-convulsant drugs. Life Sci. 39, 763-774. Rosenberg, J.S., Gilman, S.C. and Feldman, J.D. (1982) Regulation of rat B cell responses by T cell subsets and interleukin 2. J. Immunol. 129, 996-1001. Rowland, R.R.R., Tokuda, S., Weiss, G.K., Tso-Olivas, D. and Savage, D.D. (1991) Evidence of immunosuppression in the genetically epilepsy-prone rat. Life Sci. 48, 18211826.