Autoimmune effector cells part 10: Effector cells of autoimmune encephalomyelitis in healthy nonimmune rats

Journal of Neuroimmunology, 18 (1988) 315-324

315

Elsevier J'NI 00600

A u t o i m m u n e effector cells Part 10: Effector cells of autoimmune encephalomyelitis in healthy nonimmune rats

Norma S. Hayosh, Debra G. Silberg and Robert H. Swanborg Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.

(Received 20 November 1987) (Revised, received 11 January 1988) (Accepted 11 January 1988)

Key words: Autoimmuneeffector cell; Experimental allergic encephalomyelitis; Effector cell activation;

(Lewis rat, non-immune)

Summary This paper describes our ongoing investigation of the activation of effector cells of experimental allergic encephalomyelitis (EAE) from nonimmune Lewis rats by sequential culture of spleen cells (SpC) with myelin basic protein (BP) and transfer to syngeneic recipients. We show that SpC initiate the effector cell activation process, whereas thymocytes (Thy) are ineffective. Intermediary recipients of BPcultured SpC are 'primed' for EAE, but do not develop the disease; this primed state persists for at least 2 months. N o evidence was found that suppressor cells account for the failure of the intermediary recipients to develop EAE. The activation process can be inhibited by including monoclonal anti-Ia antibody in the primary culture, indicating that multiple triggering signals are involved in the activation of autoreactive T cells.

Address for correspondence: Norma S. Hayosh, Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A. Supported by research grant 2RO1NS06985-21 from the National Institutes of Health, and RG1073D7 from the National Multiple Sclerosis Society. 0165-5728/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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Introduction

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated central nervous system (CNS) disease usually induced in susceptible animals by immunization with CNS extracts or myelin basic protein (BP) administered in an appropriate adjuvant (Paterson and Swanborg, 1988). We recently described a method for the induction of effector cells of EAE from the spleens of naive (i.e., nonimmune) Lewis rats (Holda et al., 1983). Carbone et al. (1983) independently reported similar findings. This method involves the sequential culture of spleen cells (SpC) from nonimmune rats with BP, followed by transfer of the cultured cells to syngeneic recipients (Holda et al., 1983). The first culture step is thought to involve antigen processing and presentation by accessory cells (Carbone et al., 1983; Holda et al., 1983; Silberg and Swanborg, 1986). Recipients of the cells do not develop EAE, but are primed to the antigen (Silberg and Swanborg, 1986). Thus, when SpC are taken from these intermediary recipients 10-12 days later and cultured with BP, effector T lymphoblasts are generated in vitro which are fully capable of transferring EAE to secondary recipients (Silberg and Swanborg, 1986). This model is unique because it eliminates extraneous adjuvants from the system, and is of interest because it may provide insight into the steps involved in the activation of autoreactive lymphocytes. Although several stages of the activation process have been elucidated, others still require clarification. For example, it is not known why the intermediary recipients do not develop EAE. We postulated earlier that this reflects either a missing second 'signal' that amplifies the autoimmune response, or a state of natural suppression in the intermediary hosts. The possibility that suppressor T cells might inhibit EAE in these rats would be consistent with our earlier finding that suppressor T cells regulate the development of EAE in rats rendered unresponsive to BP (Swierkosz and Swanborg, 1975). Accordingly the present study was conducted in order to evaluate the immunologic status of the intermediary recipients.

Materials and methods

SpC and thymocytes (Thy) from nonimmune 8- to 10-week-old female Lewis rats (Harlan Sprague-Dawley, Indianapolis, IN) were cultured for 72 h in RPMI 1640 containing 2 p g / m l guinea pig BP, as previously described (Holda et al., 1983). The cultured cells were washed and transferred i.p. to the intermediary recipients. The intermediary recipients were given 1-1.5 x 108 cultured cells. At the appropriate time (see below) SpC a n d / o r Thy from these intermediary recipients were cultured with BP, as described above, then transferred to secondary recipients (1-1.5 x 108 cells/recipient). Monoclonal antibody (MAb, purchased from Accurate Chemical and Scientific Corp., Hicksville, NY, or Bioproducts for Science, Indianapolis, IN) was added at the beginning of the primary culture, as described (Hayosh and Swanborg, 1987).

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We utilized MAb OX3, which is specific for rat Ia antigen (McMaster and Williams, 1979) and OX39, which appears to recognize rat interleukin 2 receptors (Hayosh and Swanborg, 1987). In some experiments, rats were immunized with 25 ~g guinea pig BP in complete Freund's adjuvant (CFA), as previously described (Holda et al., 1983). Clinical EAE was graded 0 (no signs), 1 (loss of tail tonicity), 2 (paresis), or 3 (paralysis, usually accompanied by incontinence). Histologic EAE was graded on a scale of 0-4 based on the intensity of mononuclear cell infiltration in the spinal cord (Levine and Wenk, 1963). Spinal cord sections were examined without knowledge of origin.

Results

In previous studies we found that BP-cultured SpC from naive rats must reside in the intermediary recipients for 8 days before the recipients' SpC acquire the capacity to transfer EAE to secondary recipients subsequent to in vitro activation with antigen (Holda et al., 1983). Accordingly, we routinely obtained the SpC from the intermediary recipients 10-12 days post-transfer in those earlier experiments. In order to determine whether this 'primed' state persists in the intermediary recipients (i.e., whether their SpC retain the capacity to transfer EAE for a longer period of time), we transferred BP-cultured SpC to groups of intermediary recipients and sacrificed these rats at various times post-transfer. As shown in Table 1, these SpC transfer EAE to secondary recipients even when obtained 21, 25, or 66 days after

TABLE 1 EAE IN NONIMMUNE RATS: DURATION OF PRIMING OF INTERMEDIARY RECIPIENTS Cells transferred to intermediary recipients (1.5 x 10 s)

Secondary SpC culture (day) b

EAE in secondary recipients a Incidence Onset Seventy (day)

Experiment 1 BP-cultured SpC BP-cultured SpC

11 21

4/4 6/8

6.0 6.0

2.0 1.0

3/4 8/8

Experiment 2 BP-cultured SpC BP-cultured SpC

11 25

1/1 5/5

5 5.0

2 2.4

ND c 2/2

Experiment 3 BP--cuhured SpC BP-cultured SpC

11 66

2/2 3/3

7.0 7.0

1.5 1.0

ND 3/3

Histology

" 1.5 x 10S/recipient. b SpC were obtained from intermediary recipients on the day indicated (post-transfer), cultured for 72 h with BP, and transferred to secondary recipients. ¢ Not done.

318 TABLE 2 BP-PULSED THYMOCYTES DO NOT ACTIVATE EFFECTOR CELLS OF EAE EAE in secondary recipients

Cells transferred to Intermediary recipients (1.5 × 10 s )

Secondary recipients (1.5 x l0 s)

Incidence

Onset (day)

Severity

Histology

BP-cultured SpC BP-cultured Thy BP-cultured SpC BP-cultured Thy

3/3 3/3 0/8 0/8

5.0 5.0

3.0 2.3

3/3 3/3 3/3 0/3

BP-cultured SpC BP-cultured Thy BP-cultured SpC BP-cultured Thy BP-cultured Thy a

5/5 5/5 0/5 0/4 0/3

5.0 5.0

2.2 2.0

1/1 1/1 4/5 0/4 1/3

Experiment 1

BP-cultured SpC BP-cultured SpC BP-cultured Thy BP-cuhured Thy Experiment 2

BP-cultured SpC BP-cuhured SpC BP-cultured Thy BP-cultured Thy BP-cultured Thy

" Secondary culture on day 25.

the i n t e r m e d i a r y host received d o n o r cells. T h u s the i n t e r m e d i a r y recipients r e m a i n ' p r i m e d ' for at least 2 months. In the p r e s e n t s t u d y we also c o m p a r e d different p o p u l a t i o n s of a n t i g e n - p r e s e n t ing cells with respect to a b i l i t y to initiate E A E . In p r e v i o u s reports, B P - c u l t u r e d S p C o r p e r i t o n e a l e x u d a t e cells were shown to be effective ( C a r b o n e et al., 1983; H o l d a et al., 1983). However, o t h e r investigators have e m p l o y e d rat t h y m o c y t e s as a n t i g e n - p r e s e n t i n g cells for the e s t a b l i s h m e n t a n d m a i n t e n a n c e of E A E effector T cell lines ( B e n - N u n et al., 1981; V a n d e n b a r k et al., 1985). Thus, we c a r r i e d o u t a series of e x p e r i m e n t s to ascertain w h e t h e r B P - c u l t u r e d rat t h y m o c y t e s will initiate the disease process. T h e results are p r e s e n t e d in T a b l e s 2 a n d 3. A s shown in T a b l e 2, t h y m o c y t e s from naive d o n o r s are deficient with respect to a c t i v a t i n g E A E effector cells which elicit disease in s e c o n d a r y recipients. In contrast, S p C p r o v i d e a g o o d source of a n t i g e n - p r e s e n t i n g cells. However, the t h y m u s from recipients of

TABLE 3 BP-CULTURED THYMOCYTES DO NOT PRIME INTERMEDIARY RECIPIENTS FOR A C T I V E EAE Cells transferred to intermediary recipients a (5 × 10~cells)

Active EAE in intermediary recipient Incidence Onset (day)

Severity

BP-cultured SpC BP-cultured Thy No cells (control group)

9/9 12/12 3/3

1.7 2.1 2.0

8.0 13.0 13.0

a Recipients were immunized with BP-CFA 13 days post-transfer.

319 BP-cultured nonimmune SpC contains EAE effector cell precursors because BP-cultured Thy from intermediary recipients of BP-cultured SpC transfer EAE to secondary hosts (Table 2). This is in agreement with earlier findings that EAE effector cells are present in thymic tissue of rats immunized with BP and adjuvant (Hayosh and Swanborg, 1986), and recipients of activated effector cells (Naparstek et al., 1982). Further evidence that naive thymus is defective with respect to antigen presenting function is provided by the finding that intermediary recipients of BP-cultured nonimmune thymus cells are not primed for EAE (Table 3). In contrast, and in agreement with previous findings (Silberg and Swanborg, 1986), intermediary recipients of BP-cultured nonimmune SpC develop EAE in accelerated fashion when immunized with BP-CFA (Table 3). We believe this accelerated form of EAE is evidence of 'priming' of the recipients, because active disease does not develop for at least 10-12 days in nonmanipulated Lewis rats following immunization with BP-CFA. In the experiment presented in Table 3, nine recipients of BP-cultured SpC manifested clinical disease on day 8, i.e., they were primed by the BP-cultured SpC. In contrast, recipients of BP-cultured thymocytes were not primed, because they exhibited clinical EAE on day 13, the same day that the normal control rats developed EAE (Table 3). The data presented in Tables 2 and 3 indicate that rat thymocytes are not efficient antigen presenting cells for the induction of EAE in naive rats. An important question in this investigation concerns the immunologic status of the intermediary SpC recipients. As discussed previously, these rats fail to develop either clinical or histologic EAE despite the fact that they are 'primed' for disease (Table 3). Moreover, intermediary recipients that received repeated injections of BP-cultured SpC from naive rats four times, at 10-day intervals, failed to develop EAE (results not shown). Thus, repeated stimulation does not lead to disease induction. One explanation for the failure of these animals to manifest disease is that BP-cultured SpC provide only a weak antigenic stimulus which may be overcome by natural suppression (e.g., mediated by suppressor cells). To investigate this possibility, several experiments were carried out. First, low-dose irradiation of the intermediary recipients (i.e., 300 rad) prior to cell transfer does not render these rats susceptible to EAE (results not presented). A second possibility, which may also explain why Thy from nonimmune rats do not initiate the disease process (Table 2), is that the natural suppressor cells are present in thymus (Okamura and Tada, 1972). Accordingly, cell mixing experiments were performed, in which naive SpC were mixed with Thy, then cultured with BP and transferred to intermediary recipients. SpC from these rats transferred EAE to secondary recipients after in vitro culture with BP (Table 4). These experiments do not support the hypothesis that suppressor cells account for the failure of the intermediary recipients to develop EAE. To further test the suppressor cell hypothesis, experiments were carried out to ascertain whether intermediary recipients were susceptible to adoptively transferred EAE. SpC from nonimmune donor rats were cultured with BP and transferred to intermediary recipients. Thirteen days later these same rats were given BP-cultured

32O TABLE 4 THYMOCYTES DO NOT SUPPRESS ACTIVATION OF EAE EFFECTOR CELLS BY BP-CULTURED SPLEEN CELLS Cells transferred to a Intermediary recipients

EAE in secondary recipients Secondary recipients

Incidence

Onset (day)

Severity

BP-cultured SpC

5/5

5.0

2.8

BP-cultured SpC

1/2

7

1

BP--cultured SpC

2/2

7.0

1.5

Experiment 1

BP-cultured SpC + Thy b (2:1) Experiment 2

BP-cultured SpC + Thy c (2 : 1) BP-cultured SpC

a I x 10S/recipient. h Thy irradiated before culture (1500 rad). Thy not irradiated in this experiment.

S p C from o t h e r i n t e r m e d i a r y recipients (i.e., they served as s e c o n d a r y recipients). T h e s e i n t e r m e d i a r y recipients were s u s c e p t i b l e to E A E w h e n given B P - c u l t u r e d S p C f r o m o t h e r i n t e r m e d i a r y recipients, thus i n d i c a t i n g t h a t the i n t e r m e d i a r y hosts are n o t i m m u n o l o g i c a U y suppressed. T h r e e e x p e r i m e n t s were p e r f o r m e d . B P - c u l t u r e d S p C from n o r m a l rats were infused into recipients (1 x 10 8 c e l l s / r a t ) . T h i r t e e n d a y s later, these i n t e r m e d i a r y recipients were given a s e c o n d infusion of B P - c u l t u r e d SpC, but from o t h e r i n t e r m e d i a r y recipient animals. T e n of 11 recipients d e v e l o p e d clinical E A E , a n d all 11 m a n i f e s t e d histologic disease, i n d i c a t i n g that the interm e d i a r y hosts are n o t i m m u n o l o g i c a l l y s u p p r e s s e d with respect to E A E ( T a b l e 5). T o a p p r o a c h the a l t e r n a t i v e hypothesis, i.e., that a s e c o n d signal is missing in the early stages of this a u t o i m m u n e response, we i n v e s t i g a t e d the role o f Ia a n t i g e n s in the activation of E A E effector cells. Thus, S p C from naive d o n o r s were c u l t u r e d with BP in the presence of m o n o c l o n a l a n t i b o d y OX3. O X 3 recognizes Ia a n t i g e n s

TABLE 5 INTERMEDIARY RECIPIENTS ARE SUSCEPTIBLE TO ADOPTIVE EAE BY BP-CULTURED SpC FROM OTHER INTERMEDIARY RECIPIENTS SpC transferred to intermediary recipients from

EAE in intermediary recipients Clinical Histologic

Naive donors Other intermediary recipients a

0/7 10/11

ND b 11/11

a SpC were obtained from intermediary recipients 13 days post-transfer, cultured with BP then transferred to other intermediary recipients. Each rat received 1.5 × 10 8 BP-cultured SpC from another intermediary recipient. b Not done.

321 TABLE 6 MONOCLONAL ANTIBODY OX3 BLOCKS EAE EFFECTOR CELL ACTIVATION WHEN INCLUDED IN NAIVE SpC CULTURE Cells transferred to Intermediary recipients (1.4x 108) SpC + BP+ OX3 " SpC + BP + OX39 b SpC + BP (control)

Secondary recipients (1.4x 108) BP-cuhured SpC BP-cultured SpC BP-cultured SpC

EAE in secondaryrecipients Incidence 1/7 1/1 8/10

a 2 ~tg/ml OX3 (anti-la). b 5/~g/ml OX39 (anti-interleukin2 receptor). on rat lymphocytes (McMaster and Williams, 1979). As shown in Table 6, when OX3 was included in the primary SpC cultures, the activation of effector cells of EAE was inhibited, as reflected by failure to transfer EAE to the secondary recipients. That inhibition is specific is indicated by the finding that the inclusion of an irrelevant antibody (OX39, specific for interleukin 2 receptors) in the primary culture did not result in abrogation of adoptive transfer of EAE (Table 6).

Discussion

The present study was carried out to further elucidate the mechanism of EAE effector cell induction in nonimmune rats. This model involves sequential culture of donor spleen ceils with BP, and transfer to syngeneic recipient rats (Holda et al., 1983; Silberg and Swanborg, 1986). Thus, when SpC from nonimmune donors are cultured with antigen and transferred to intermediary recipients, the SpC from these intermediary hosts acquire the capacity to transfer EAE to secondary recipients subsequent to in vitro culture with BP (Carbone et al., 1983; Holda et al., 1983; Silberg and Swanborg, 1986). A crucial, and as-yet unresolved question relates to why the intermediary recipients do not eventually develop EAE. It is clear from the data presented in Table 1 that these intermediary recipients remain 'primed' for at least 2 months, because SpC are still capable of transferring EAE following activation with BP when taken from intermediary recipients on day 66. However, these intermediary hosts do not manifest either clinical or histological signs of EAE (Holda et al., 1983). We have assumed that the initial event in EAE effector cell activation, which occurs in the first in vitro culture step (i.e., culture of naive SpC with antigen) reflects antigen processing and presentation, because this step is radioresistant (Silberg and Swanborg, 1986), and can also be achieved with BP-pulsed peritoneal macrophages (Carbone et al., 1983; Holda et al., 1983). However, this may be an oversimplification, because if the first culture step serves merely to provide a source

322 of antigen-presenting accessory cells to the intermediary recipient, BP-pulsed thymocytes ought to initiate the autoimmune response. Thymocytes reportedly function perfectly well as accessory cells to maintain BP-specific rat T cell lines (Ben-Nun et al., 1981; Vandenbark et al., 1985). In the present study, BP-cultured Thy were ineffective with respect to the induction of EAE (Tables 2 and 3). Since others have successfully used rat Thy as accessory cells, this discrepancy suggests that the failure to elicit EAE in naive rats with BP-pulsed Thy cannot be attributed to inefficient antigen presentation. Rather, this finding may suggest that Thy lack some other attribute which is required for complete induction of the autoimmune response. One might speculate that another cell type may be missing from the thymus; this cell might be involved in the induction of EAE by virtue of providing a second signal necessary for the development of the autoimmune response. In this regard, it should be noted that Sakai et al. (1986) found that Thy from mice with chronic relapsing EAE failed to proliferate in response to BP, whereas SpC from the same animals responded to the antigen. It has been postulated that two signals are required for the activation of EAE effector cells (Holda et al., 1983; Mannie et al., 1987), one provided by antigen and the other by lymphokines generated in culture. We favor this hypothesis, and have found no evidence to support the alternative hypothesis, i.e., that suppressor cells inhibit the development of EAE in the intermediary recipients. For example, in cell-mixing experiments, secondary recipients of SpC from intermediary recipients that were given BP-cultured SpC + Thy (2 : 1) developed adoptive EAE. Moreover, transfer of BP-activated SpC from intermediary recipients to other groups of intermediary recipients (i.e., rats that had previously received BP-cultured SpC from naive donors) elicited EAE in these hosts (Table 5). Had a state of suppression existed in these rats, one would not have expected them to develop this autoimmnne disease. Furthermore, low-dose irradiation of intermediary recipients, which should have inactivated suppressor cells (Anderson and Warner, 1976) did not lead to EAE induction in these rats. Thus, the data do not lend support to the suppressor cell hypothesis. The most likely alternative is that a required 'second signal', which may amplify antigen presentation, is missing in the intermediary recipients. The finding that thymocytes fail to induce the autoimmune response suggests that the source of this 'second signal' is not to be found in the thymus, but is present in the spleen. Although the nature of this putative second signal is unknown, it is likely that soluble mediators derived from lymphocytes or macrophage/monocytes are important in this response. Interleukin 1 (IL-1), released from accessory cells, is necessary for the activation of EAE effector cells from BP-CFA-immunized rats (Killen and Swanborg, 1982; Marmie et al., 1987). Subsequently, interleukin 2 (IL-2) is released from T lymphocytes (Ortiz-Ortiz and Weigle, 1982). IL-2 probably amplifies the autoimmune response, as demonstrated by the finding that monoclonal antibodies specific for IL-2 receptors inhibit the activation of EAE effector cells (Hayosh and Swanborg, 1987; Sedgwick et al., 1987). Interferon-~, (IFN-~,) may also contribute to this second signal. IFN-~, appears to play a crucial role in the development of autoimmune diseases by inducing the expression of class II major histocompatibility

323

antigens in situ (McCarron et al., 1985), and we recently demonstrated that IFN-~, is produced by EAE effector cells (McDonald and Swanborg, 1988). Although the nature of the second signal remains to be defined, the finding that monoclonal antibody OX3 inhibited EAE effector cell activation suggests that class II antigens are important in the inductive phase of this autoimmune response. Studies are in progress to further clarify the steps involved in the activation of effector cells of EAE. In conclusion, our findings reveal that EAE effector cells or their precursors exist in healthy Lewis rats without causing disease. The elucidation of the mechanisms responsible for the regulation of these effector cells should advance our understanding of immunological homeostasis, and may have relevance to multiple sclerosis, a disease in which immunoregulatory defects have been found (for review, see Ratine (Ed.), 1984).

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