T cells in the spinal cord in experimental autoimmune encephalomyelitis are matrix adherent and secrete tumor necrosis factor alpha

Journal ofNeuroimmunology, 37 (1992) 161-166

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© 1992 Elsevier Science Publishers B.V. AII rights reserved 0165-5728/92/$05.00 JNI 02158

Short communication

T cells in the spinal cord in experimental autoimmune encephalomyelitis are matrix adherent and secrete tumor necrosis factor alpha Rami Hershkoviz, Felix Mor, Dalia Gilat, Irun R. C o h e n and O f e r Lider Department of Cell Biology, The Weizmann Institute of Science, Rehocot 76100, Israel (Received 11 October 1991) (Revised, received 20 November 1991) (Accepted 21 November 1991)

Key words." T cell; Extracellular matrix; Tumor necrosis factor-a; Spinal cord; Experimental autoimmune encephalomyelitis

Summary We examined T cells isolated from an autoimmune tissue lesion and from lymphoid organs for their ability to secrete tumor necrosis factor-a (TNF-a) and to adhere to extracellular matrix (ECM) proteins. CD4 ÷ T ceils were obtained from spleens, popliteal lymph nodes, and spinal cords of Lewis rats that had been immunized with myelin basic protein (MBP) to induce experimental autoimmune encephalomyelitis (EAE). We now report that, irrespective of whether or not the T cells were activated with MBP or the T cell mitogen concanavalin A (ConA), the T cells isolated from the spinal cord lesions secreted greater amounts of TNF-a and adhered better to ECM than did T cells from the draining lymph node. Thus, the lesions of EAE concentrate a subpopulation of CD4 ÷ T cells with enhanced ability to interact with blood vessel wall components and to secrete TNF-a.

Introduction In the absence of disease, migration of T cells into and through the central nervous system (CNS) is limited. However, in the case of multiple

Correspondence to: Dr. O. Lider, The Department of Cell Biology, R326, The Weizmann Institute of Science, P.O. Box 26, Rehovot 76100, Israel. O. Lider is the recipient of the Allon scholarship and incumbent of the Weizmann League Career Development Chair in Children's Diseases. I.R. Cohen is the incumbent of the Mauerberger Professorial Chair in Immunology.

sclerosis or experimental autoimmune encephalomyelitis (EAE), T cells expressing various antigenic specificities and restricted numbers of T cell receptor (TCR) V gene segments can be found in the CNS (Wekerle et al., 1986; Hailer et al., 1988; Naparstek et al., 1984; Wucherpfenning et ai., 1990). The idea behind the present study was to investigate whether the T ceils accumulating in the autoimmune lesion differed functionally from the T cells present in the draining lymph node or spleen. We studied two functions: adherence to extracellular matrix (ECM) protein components and secretion of tumor necrosis factor-a (TNF-a).

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TNF-a, because of its diverse and far-reaching effects on the inflammatory process, can be viewed as one of the most powerful mediators of inflammation (Beutler and Cerami, 1989; Ruddle et al., 199(I). The adhesion of T cells to ECM protein components could contribute to their accumulation in tissues (Springer, 1990; Shimizu and Shaw, 1991). We now report that T cells isolated from spinal cords were more adherent to ECM components and produced more TNF-a than did equal numbers of T cells isolated from the draining lymph nodes or spleens of rats suffering from EAE. These characteristics of the spinal cord T cells were evident irrespective of whether or not the T cells were activated by MBP or the mitogen concanavalin A (ConA). Thus, in addition to antigen-specific T cells, the inflammatory lesion selectively concentrates T cells with particular functional properties.

Materials and methods

Rats. Inbred female Lewis rats were supplied by the Animal Breeding Center of the Weizmann Institute and were used at 2-3 months of age. Antigens. Guinea pig myelin basic protein (MBP) was prepared as previously described (Diebler et al., 1972). Synthetic peptide residue 72-89 was purchased from Biosearch, CA, USA. Induction of EAE. Disease was induced by injecting hind footpads of recipient rats as previously described (Beraud et al., 1989). Preparation of cell suspensions. Twelve days after induction of EAE, rats were sacrificed and single-cell suspensions were prepared from lymph nodes, spleens or thymuses by pressing the organs through a fine wire mesh. Spinal cord lymphocytes were obtained by gentle grinding of spinal cord tissue with a 15 ml Dounce tissue grinder (Wheaton 357544, N J, USA) in 10 ml of phosphate-buffered saline (PBS). The homogenate was then subjected to 2-3 cycles of Ficoll gradient separation (Pharmacia, Sweden). The resulting cells were then cultured in microtiter wells and the non-adherent cells were recovered from the dishes and applied on pre-warmed nylon-wool columns. A single-cell suspension was made and

the cells were exposed to the following monoclonal antibodies (mAbs): OX-6, anti-major histocompatibility complex (MHC)-II-Ia antibodies diluted 1:500; OX-8 anti-suppressor/cytotoxic T cell antibody diluted 1:350; OX-33 anti-rat B cell diluted I:4(KI, and OX-41 anti-rat dendritic cells and macrophages diluted 1:450 (all mAbs were purchased from Serotec, UK). After 30 min at 4°C, the cells were washed and mixed with magnetic beads (Dynal P-450, Norway) coupled to goat anti-mouse IgG antibodies for an additional 30 min at 4°C. The purified CD4 + cells that did not adhere to the antibody-coated beads were collected. The recovered cells, above 90% CD3+CD4 + T cells, proven by phcnotype analysis of the cells using a FACScan, were used. Limiting dilution analysis. The lymphocytes obtained from the various sources were seeded in 96-well, round-bottom microtiter plates at decreasing concentrations from 4 × 103 cells per well to one cell per well, in doubling dilutions. The cells were seeded in 200 izl Dulbecco's modified Eagle's medium (DMEM) supplemented with 2-mercaptoethanol (2-ME) (5 × 10 5 M), eglutamine (2 mM), sodium pyruvate (1 mM), antibiotics and 10% fetal calf serum (FCS), and T cell growth factor. Irradiated thymocytes (2500 R) were added as feeder cells (10 s per well). Cultures were performed in the absence or presence of MBP (10 /xg/ml) or ConA (1.2 g g / m l ) . Twenty-four wells were seeded at each cell concentration; 20 wells with MBP or ConA and four wells as controls without either. Plates were incubated for 7 days in 7% CO 2 at 37°C. The uptake of [3H]thymidine was measured in the last 18 h of culture. A positive well was scored by a visible cellular aggregation and thymidine uptake exceeding the mean plus 2 standard deviations of the background wells cultured without antigens or ConA. Percent negative wells was plotted against cell concentration on a semilog plot. The cell number corresponding to 37% negative wells was considered to represent the frequency of the T cells responding to the antigen (Lefkovits and Waldman, 1979). TNF secretion assays. 250,000 T cells were activated with 20/.Lg/ml MBP or 2 / x g / m l ConA in RPMI medium supplemented with 0.1% bovine serum albumin (BSA) in round-bottom 96-well

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plates (Costar). The plates were incubated at 37°C in a humidified incubator for 6 h. Subsequently, the contents of the wells ( 3 - 6 wells per experimental group) was collected, centrifuged, and the media were assayed for TNF secretion done as previously described (Wallach, 1986). To examine the molecule assayed as TNF-a, mAbs to TNF-a were included in some culture wells of HeLa cells and were found to inhibit the effect of TNF-a (data not included). Adhesion assays. ECM-coated wells were prepared as previously described (Lider et al., 1989). BSA, laminin (LN), fibronectin (FN), FN peptides GRGDSPK or control GRGESP were purchased from Sigma. Anti-FN (specific for the cell-attachment site of FN) mAbs were purchased from Boehringer-Mannheim (Germany), and

polyclonal antibody anti-LN were purchased from Bio-Makor (Israel). The procedure of the adhesion experiments was designed and performed as previously described (Shimizu et al., 1990; Lider et al., 1991), except that for the MBP-induced activation of the T ceils which was done in the presence of irradiated thymocytes as antigen-presenting cells (APC). Since only the lymphocytes were radioactively labeled, the levels of adhesion represent those of the lymphocytes and the APC.

Results and discussion

The ability of T cells to interact with blood vessel wall components such as endothelial cells,

T A B L E 1A SPLEEN, SPINAL C O R D A N D L Y M P H N O D E T C E L L A D H E S I O N T O ECM A N D ECM P R O T E I N C O M P O N E N T S T cells were isolated from draining lymph nodes, spleen and spinal cords of M B P / c o m p l e t e Freund's adjuvant (CFA)-immunized Lewis rats. The cells were then either activated with MBP (in the presence of irradiated thymocytes) or C o n A or left intact and the ability of these labeled lymphocytes to adhere to immobilized BSA, LN, FN or ECM was examined (Table 1A). Some wells were pretreated with anti-LN or anti-FN antibodies (Table 1B), and, where indicated, the ConA-activated T cells were pre-treated with either G R G D S P K or G R D E S P peptides and their adhesive properties were then examined. Group

1 2 3 4 5 6 7 8 9

Source of T cells

Activation of T cells

% T cell adhesion ( ± SD) to BSA FN

LN

ECM

Spleen

None ConA MBP None ConA MBP None ConA MBP

3 4 3 3 5 4 4 3 2

4+2 22+4 16+3 4±2 27+3 22±2 20 + 4 63_+4 57 + 4

5±1 29+5 16:t:4 5±1 30:t:4 20+4 46 ± 6 79±6 67 ± 3

Lymph nodes

Spinal cord

± 2 ± 1 5:1 ± 2 ± 3 + 3 ± 1 ± 2 ± 1

4+1 19±2 15+3 5±2 21 ± 3 16±4 23 ::1:4 60:t:3 53 ± 5

T A B L E IB SPINAL C O R D A N D L Y M P H N O D E T CELLS A D H E R E T O T H E FN C O M P O N E N T O F T H E ECM VIA T H E I R INTEGRIN RECEPTORS Group

Inhibitor

% T cell adhesion to (% inhibition) Spinal cord T cells

1 2 3 4 5

None GRGDSPK GRGESP Anti-FN m A b Anti-LN antibody

Lymph node T cells

120 kDa of FN

LN

120 kDa of FN

LN

50 + 4 7+ 3 52+3 11 + 2 48 ± 2

50 :t: 3 48 ± 3 53_+3 47 + 5 12 + 4

31 + 4 12 -1- 2 33_+2 10 ± 3 33 _+ 4

25 ± 5 24 + 5 26±3 23:1:4 10 + 2

(84) (0) (78) (0)

(0) (0) (0) (76)

(62) (0) (68) (0)

(0) (0) (0) (60)

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subendothelial ECM, and interstitial matrix is pivotal in regulating cell traffic and migration (Springer, 1990). It has been shown that "F cell adhesion to protein components of the ECM, mediated by integrin receptors, is augmented with cell activation (Shimizu ct al., 1990), and that activated anti-MBP T cells accumulate in the CNS in E A E (Naparstek et al., 1983). We therefore examined the ability of T cells isolated from various organs of rats with E A E to adhere to components of the ECM. We isolated T cells from spinal cords, spleens, and draining lymph nodes of the rats and compared their adhesive properties to FN, LN and ECM with or without additional activation in vitro or with either ConA or MBP. The results shown in Table I A demonstrate that T cells isolated from spinal cords adhered to the indicated substrates; their level of adhesion was 5 - 9 times higher than that of the T cells isolated from the lymph nodes or spleens. Thus, it appears that an enriched subpopulation of cells is present in the autoimmune lesion site in the CNS which are capable of interacting with blood vessel wall components in the absence of exogenous stimulus. T cell adhesion levels to immobilized BSA, which served as control protein, were very low. It was possible that a higher proportion of spinal cord T cells adhered to the ECM proteins because the lesion was enriched in activated T cells. We therefore activated in vitro the T cells using ConA or MBP. Table 2 shows that thc

FABLE 2 F R E Q U E N C Y ANALYSIS O F A N T I - M B P A N D ('onAR E A C T I V E T CELLS I S O L A T E D F R O M I..YMPII N O D E S A N D SPINAL C O R D S O F RATS W I T H E A E CD4 + T cells were isolated from spleens, lymph nodes and spinal cords of Lewis rats which had been immunized with MBP in complete Freund's adjuvant 12 days before. The frequencies of reactive T cells were assayed by limiting dilution. T cells isolated

Frequency of reacting T cells (%)

from

MBP

ConA

Spleens Lymph nodes Spinal cord

0.1 0.4 4

ND 100 50

lymph nodc cells responded to ConA with a higher frequency (100%:) than did the spinal cord T cells (50%), but that the spinal cord "I" cells wcrc 10-fold enriched in MBP-responsive T cell (4c/, vs. 0.4%). Nevertheless, even after activation by ConA as well as by MBP, a higher proportion of spinal cord "I" cells adhered to the substrates. Thus, more T cells from the E A E lesions adhered compared to the peripheral T cells irrespective of activation in vitro. The frequencies of MBP-reactive T cells we found appear to be higher than those reported previously. We propose several explanations for this finding: (a) other researchers may have missed the high frequency of autoreactive cells because they did not test concentrations of cells bellow 8-1{} 2 per well; and (b) limiting dilution analysis can be influenced greatly by differences in culture conditions. Indeed, our culture medium was enriched with growth factors from thc onset of the cell culture while others added intcrlcukin-2 only on day 4 (F. Mor et al., in preparation). Do the T cells present in the lesion recognize and interact with ECM proteins by means of conventional integrin receptors or is the added adherence due to novel receptors? The results shown in Table 1B indicate that the adhesion of lymph node T cells to both FN and I_,N was markedly enhanced with cell activation, and that T cell adhesion to FN was inhibited if the immobilized FN was pre-treated with antiFN mAbs, but not with anti-LN antibodies, t,ikewise, activated T cell adhesion to LN was inhibited by anti-LN antibodies, but not by anti-FN mAbs. It has been demonstrated that lymphoid cells recognize and attach to bound FN by VLA receptors which interact with the tripeptidc ArgGly-Asp ( R G D ) (Ruoslahti and Pierschbacher, 1987). Therefore wc tested the effect of soluble G R G D S P K and of the control peptide containing the Arg-Gly-Glu ( R G E ) sequence on T cell adhesion. The results shown in Table 1B shows that T cell adhesion was inhibited by R G D but not by the R G E peptide. Thus, the property of adhesion to ECM and ECM protein components of the various T cell tested is integrin mediated. Thus, the augmented adherence of lesional T cells appears to depend on the conventional integrin receptors found on lymph node T cells.

165 TABLE 3 TNF-a SECRETION RATS W I T H E A E

BY T CELLS I S O L A T E D F R O M

Purified T cells obtained from spleens, popliteal lymph nodes or spinal cords of rats with E A E were seeded in microtiter wells in the absence or presence of MBP or ConA, in the absence of antigen-presenting cells. The supernatants were collected and tested for the presence of TNF-a. The results shown here represent one of three experiments which yielded essentially the same results. Group

T cells isolated from

in vitro activation

T N F - a secretion ( p g / m l _+SD)

1 2 3 4 5 6 7 8 9

Spleen

None ConA MBP None ConA MBP None ConA MBP

30.-2. 6 250 +_35 260 _-2-3O 25_+ 5 300 + 35 330 _+45 155 +_ 10 790 + 50 680 --2.55

Lymph nodes

Spinal cord

Another pivotal feature of reactivity of immune cells is their ability to secrete inflammatory cytokines. We chose to examine the ability of the EAE T ceils to secrete T N F - a since TNF-a, because of its diverse and far-reaching effects on the inflammatory process, can be viewed as one of the most powerful mediators of inflammation (Sobel et al., 1986; Male et al., 1990; Osborn, 1990). Accordingly, CD4 ÷ T ceils were purified from the mononuclear leukocytes obtained from spleens, lymph nodes and spinal cords of paralyzed rats. The CD4 ÷ T cells were then cultured in vitro in the absence or presence of either MBP or ConA. Culture supernatants were collected and tested for the presence of TNF. The results, shown in Table 3, indicate that spinal cord T ceils secreted relatively higher amounts of T N F compared to those secreted by lymph node or spleen cells. The high levels of T N F were inhibited by a mAb to T N F - a indicating that the molecule assayed as T N F was T N F - a (data not shown). Upon activation with MBP or ConA, CD4 ÷ T ceils isolated from the spleens or lymph nodes also secreted TNF-a. However, the T ceils isolated from the spinal cords were found to secrete even a greater amount of TNF-a.

The results of the present study indicate that T ceils accumulating in the spinal cord lesions of rats suffering from E A E are capable of augmented adherence to ECM and increased secretion of TNF-a. Activating the T ceils with the mitogen ConA or with the specific antigen MBP augmented adherence and T N F - a secretion; nevertheless, the peripheral T cells did not equal the performance of the lesional T ceils. Thus, the relative number of activated cells cannot explain the augmented performance of the spinal cord T cells. Moreover, the low percentage of MBP-reactive T cells in the CNS cannot explain the augmented number of matrix-adherent and TNF-asecreting T cells. We suggest that the level of T cell adhesion to ECM and its protein components, as well as their ability to secrete higher amounts of TNF-a, reflect the migratory ability of both the antigen-specific and non-antigenspecific T cells into the CNS. Thus, the infiltrating T cells are enriched in a subpopulation of T cells intrinsically more active in interacting with ECM components and inflammation; the T cells in the lesion are not a random sample of the T cells present in the lymph node and in the circulation. This suggests that specific functions may be performed even by the T cells that do not recognize the specific antigen yet accumulate in the autoimmune lesion.

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