Immunology 1977 33 621
Reactive and non-reactive lymphocytes in experimental allergic encephalomyelitis II.
CHARACTERISTICS OF PERITONEAL LYMPHOCYTES RESPONDING TO ANTIGEN
A. I. LEVINSON, R. P. LISAK, B. ZWEIMAN & L. D. WILKERSON Division of Allergy and Immunology, Department of Medicine and the Department of Neurology, School of Medicine, University of Pennsylvania; University of Pennsylvania and Wistar Institute Multiple Sclerosis Research Center, U.S.A.
Received 4 November 1976; acceptedfor publication 9 March 1977
Summary. Myelin basic protein (BP), induces an increased in vitro proliferative response of peritoneal exudate cells (PEC), compared with that given by peripheral blood lymphocytes (PBL), in guinea-pigs with experimental allergic encephalomyelitis (EAE). This response is determined by the nature of the lymphocytes in such exudates. We have also found that: (a) the majority of peritoneal lymphocytes from non-sensitized animals are E-rosetting (T cells) (59%) or null cells (>40%) with EAC-rosetting (B cells) comprising 1% or less of cells; (b) both T cells and null cells respond equally to BP as determined by a technique combining rosette-formation and autoradiography; (c) the increased in vitro peritoneal lymphocyte response in animals with EAE cannot be explained solely by the number of null cells since peripheral blood and lymph nodes also contain appreciable numbers of null cells.
to the sensitizing antigen than are comparable numbers of lymphoid cells from other compartments
(Rosenstreich, Blake & Rosenthal, 1971; Rosenthal, Rosenstreich, Davie & Blake, 1972). Studies from this laboratory have demonstrated an increased reactivity of peritoneal lymphocytes of animals with experimental allergic encephalomyelitis (EAE) to homologous myelin basic protein (GP-BP), the antigen that induces this experimental autoimmune disease (Lisak & Zweiman, 1973; Lisak, Zweiman, Kies & Driscoll, 1975; Lisak, Zweiman & Levinson, 1976). The reasons for the enhanced in vitro reactivity are not well-defined, since both peritoneal macrophages (Rosenstreich et al., 1971) and the lymphocytes found in peritoneal exudates (Rosenstreich et al., 1971; Lisak et al., 1976) could be responsible. In recent studies we concluded that it was unlikely that the greater reactivity of peritoneal lymphocytes than blood lymphocytes to GP-BP was attributable solely to the macrophages in the peritoneal exudate cell population (Lisak et al., 1976). It was conceivable that lymphocytes particularly responsive to GP-BP might contribute to the increased reactivity of the peritoneal exudate cell population. Accordingly, we have characterized the peritoneal lymphocytes that proliferate in vitro, identifying
INTRODUCTION
Peritoneal exudate inflammatory cell populations include lymphocytes that are more reactive in vitro Correspondence: Dr R. P. Lisak, Department of Neurology, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, U.S.A. B
621
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A. I. Levinson et al.
T and B lymphocytes by resetting techniques in combination with autoradiographic assays of in vitro proliferation induced by GP-BP (Elfenbein, Shevach & Green, 1972).
MATERIALS AND METHODS
Immunological characteristics of cells Preparation of tissue lymphocyte suspensions. (a) Thymus and lymph node. Unsensitized male Hartley guinea-pigs, weighing 400-500 g were injected i.p. with sterile mineral oil (Marcol, Humble Oil, Houston, Texas) Three days later, these animals were killed and the cervical lymph nodes and thymuses were removed. After thorough washing in Hanks's balanced salt (HBSS, GIBCO, Grand Island, New York), single cell suspensions were prepared by teasing apart the tissues, pressing them through a 30-gauge stainless steel mesh screen, and excluding residual debris by gravity sedimentation. Cells were suspended at a concentration of 2 x 106/ml in HBSS. (b) Peritoneal exudate lymphocytes. Cells were obtained by peritoneal lavage 3 days after the i.p. injection of sterile mineral oil, washed twice with HBSS and incubated for 30 min on a glass wool, nylon wool column. The column was prepared by packing I 0 g of nylon wool (Fenwall Laboratories, Division of Travenol Labs, Inc., Morton Grove, Illinois) into a 10-ml vol. in the bottom of a 50-ml plastic syringe followed by glass wool (Fisher Scientific, King of Prussia, Pennsylvania) occupying a further 10-ml vol. Both materials were pre-washed with warmed HBSS containing 10 per cent foetal calf serum (FCS) v/v and the column was incubated at 370 prior to use. The cells eluted from the column consisted of 95-98%4 small- and medium-sized lymphocytes; the remainder were cells capable of phagocytozing oil and/or latex particles. The eluted cells were washed and resuspended in HBSS (10% FCS) at a concentration of 2 x 106 cells/ml. Cell viability was consistently greater than 98% judged by trypan blue exclusion. (c) Peripheral blood lymphocytes (PBL). Mononuclear cell suspensions were prepared from heparinized blood by centrifugation over FicollHypaque density gradients (Boyum, 1968). Cells at the interface consisted of 5-20 per cent monocytes (phagocytic mononuclear cells identified after incubation with 1 1 jpm latex particles) and lympho-
cytes. The cells were suspended at a concentration of 2 x 106 cells/ml. Determination of cell-surface characteristics. (a) E rosettes. 0-05 ml of a concentrated suspension of washed latex particles (I 1 pam diameter, Coulter Electronics, Hialeah, Florida) was added to duplicate tubes containing 0-2 ml of either thymus, lymph node, PBL or PEL suspensions. Following incubation for 45 min at 370 on a mechanical rotator the cells were washed and the supernatants replaced with 0-2 ml fresh HBSS. Equal volumes of a I % solution of freshly drawn rabbit red blood cells (RRBC) were added to the resuspended cells. Following incubation on a mechanical rotator for 15 min at 370 the cells were centrifuged at 200 g at 40 for 5 min and kept on ice for 1 h. Subsequently the pellets were gently resuspended and the numbers of rosettes were counted. Lymphocytes with three or more adherent RRBC were scored as rosettes. Cells containing at least two latex particles were considered to be monocyte/macrophages and were excluded from the counts. Two hundred mononuclear cells were counted routinely. (b) EAC rosettes. Equal volumes of lymphoid cells prepared as above, sheep red blood cells
(SRBC) sensitized with IgM haemolysin (Cordis Laboratories, Miami, Florida) and mouse complement (AKR serum, Jackson Laboratories, Bar Harbor, Maine) were incubated on a mechanical rotator for 30 min at 37°. The suspensions were then examined for the percentage of latex-negative cells which were rosette positive. Identification of BP-reactive lymphocytes in lymphocyte-enriched peritoneal exudates of EAE animals Animal sensitization. Male Hartley albino guineapigs (450-700 g) were injected i.d. over the sternum on day 0 with 100 ug of guinea-pig basic protein (GP-BP), prepared by the method of Diebler, Martenson & Kies (1972), in Freund's complete adjuvant (containing 100 ug M. tuberculosis H37R., Difco Laboratories, Detroit, Michigan, in 0-1 ml). This method of sensitization has consistently induced EAE in our laboratory (Lisak & Zweiman, 1974a; Lisak & Zweiman, 1974b; Lisak et al., 1975). On day 10, 30 ml of sterile mineral oil was injected i.p. On day 13, peritoneal cavities were lavaged and exudate cells were harvested as previously described (Lisak et al., 1974a; Lisak et al., 1976).
Reactive and non-reactive lymphocytes
Cell separation and culture techniques. Cells were washed twice in HBSS and passed over a column of pre-washed coiled cotton (Type A, Fisher Scientific) in order to enrich the lymphocyte population. The cells eluted from the column consisted of 50-80% small and medium lymphocytes, 5-10% polymorphonuclear cells, and 30-50% macrophages. The latter were identified by their ability to ingest oil and/or latex particles. These cotton filtered peritoneal exudate cells (PECcF) were suspended in Spinnermodified Eagle's Minimal Essential Medium (MEM-S, GIBCO) supplemented with penicillin, streptomycin, glutamine and FCS (l0%Yo v/v) and cultured for 4 days at a concentration of 1 x 106 lymphocytes/ml at 370 in 5% C02/air. To multiple replicate cultures were added either: (1) GP-BP in the final concentration of 25 ug/ml, or (2) additional culture medium. Following a terminal 18-h pulse of tritiated thymidine, the contents of three tubes of each replicate group were processed for the assay of isotope uptake by scintillation counting. The degree of proliferation was expressed as a stimulation index (SI) where: - mean
c.p.m. of GP-BP-stimulated cultures mean c.p.m. of control cultures
Rosetting-autoradiography assay. The remaining replicate cultures were assayed by a combination of rosetting and autoradiography. Replicate cultures were pooled; the cells were washed and E or EAC rosettes were formed. To preserve the integrity of the rosettes, glutaraldehyde in FCS (previously absorbed with RRBC) was added to the rosetting suspensions at a final concentration of 2-5% for E rosettes and 10% for EAC rosettes. Preliminary studies in our laboratory indicated that this modification did not significantly alter the percentage of rosettes as compared with our standard method. Rosetting suspensions were transferred to gelatin-coated glass slides and allowed to dry before final fixation in methanol. The slides were dipped in Kodak NTB-2 emulsion (Kodak, Rochester, New York) and incubated for 7 days at 40 for autoradiography as previously described (Phillips & Zweiman, 1970). After development and staining with Giemsa solution, at least 400 cells were examined for the percentages of E-rosetting lymphocytes, the percentage of labelled cells, the percentage of rosette cells that were labelled, and the percentage of labelled cells that formed rosettes. All cells that had
623
1ooL 80 Vn -
2 60 2 20
0
E EAC N
Thymus
E EAC N Lymph node
E EAC N
E EAC N
PBL
PELt
E EAC N PEC t
Figure 1. Lymphocyte sub-populations in non-sensitized guinea pigs. Mean ± s.e.m.; macrophage/monocytes excluded from the counts; * PBL =peripheral blood mononuclear cells; t PEL = peritoneal exudate lymphocytes; * PEC = unfiltered peritoneal exudate cells. E = E-rosetting (T) cells; EAC = EAC-rosetting (B) cells; N = null cells.
ingested at least two latex particles, presumably macrophages, were excluded from the counts.
RESULTS Surface characteristics of lymphocytes The prevalence of E-and EAC-rosetting cells in the PEL of unsensitized animals is indicated in Fig. 1. The majority (59%) of the PEL were T cells forming E rosettes; no EAC-rosetting cells were present. However, over 40% on average, of the PEL formed neither E nor EAC rosettes; hence, the term 'null cells' was applied. To rule out the possibility that these results were due to insensitive rosetting techniques, comparative studies of thymus, lymph node cells and PBL were made using the same assays. The large majority of thymocytes formed E rosettes (mean=92%) but not EAC rosettes. The mean percentages of E- and EACrosetting cells in the lymph node lymphocyte population were 55% and 31% respectively. The PBL populations contained a mean of 60% E and 13% EAC rosettes. These findings are similar to those reported elsewhere (Staedecker, Bishop & Wortis, 1973; Wilson & Coombs, 1973; Sandberg, Soder & Ernstrom, 1976) and attest to the sensitivity of the techniques. Thus the percentage of null cells was significantly higher in the normal PEL than in the thymus and lymph node cells, but not the PBL populations. To determine whether the cell enrichment
A. I. Levinson et al.
624
Table 1. Contributions of T and null cells to the proliferative response to GP-BP* in animals with EAE
Experiment
SIt
Per cent labelled cells
Per cent E-rosetting cells
1 2 3 4 5 6
8 26 16 17 50 6
71 40 26 33 43 9
45 35 23 31 45 18
Per cent labelled cells Per cent E rosette that form E rosettes cells that are labelled 37 26 27 27 50 48
60 29 23 28 37 23
* GP-BP = Guinea-pig basic protein. t SI=mean c.p.m. of GP-BP stimulated cultures mean c.p.m. of control cultures
procedure for obtaining PEL may have been responsible in part for the prominence of null cells in this population, additional rosetting studies were performed on unfiltered peritoneal exudate cells (PEC). These cells were incubated with latex beads before rosetting was attempted so that phagocytic cells could be excluded. As seen in Fig. 1, the percentage of lymphocytes which rosetted with neither E nor EAC preparations was higher in PEC than that found in PEL (mean percentage of null cells = 61%). This suggests that column filtration of the peritoneal exudate cells did not selectively enrich the null cell
population. In vitro proliferative response to BP We determined which cells within the peritoneal exudate cell population of animals with EAE proliferated in vitro in response to GP-BP. The column fractionation used to prepare PEL led to considerable reduction in cell yield (Rosenstreich & Rosenthal, 1974; Lisak et al., 1976), so that cell numbers were insufficient for the assays combining rosetting and autoradiography. The pooling of PEL from several inbred animals (Rosenstreich et al., 1971) was not feasible in the present experiments with outbred Hartley guinea-pigs. Therefore, an alternative method was used. Peritoneal exudate cells passed over coiled cotton columns (PECCF), were composed of 30-50% macrophages, 40-50% lymphocytes and 5-10% granulocytes. Of the lymphocytes, 10-40% were T lymphocytes and 55-70% were null cells. No EACrosetting cells were seen.
We have previously found that PECCF from EAE animals proliferated briskly when cultured with GP-BP as measured by scintillation spectrometry (Lisak et al., 1973; Lisak et al., 1976). In the present study PECcF cultured with and without GP-BP were assayed by a combination of rosetting and autoradiography and the results were compared. It can be seen (Table 1) that GP-BP induced the proliferation of both T cells and null cells. Fig. 2 illustrates an example of a labelled T (E rosetting) and a labelled non-E-rosetting cell (null cell). As expected, no EAC-rosetting cells were found in these cultures. The phagocytic cells remaining in the cultures were neither labelled nor rosette forming. The proliferative response measured by autoradiography was confirmed by concomitant scintillation counting studies (SI ranging from 6-50). In contrast, no significant proliferation was seen in unstimulated cultures (
Reactive and non-reactive lymphocytes in experimental allergic encephalomyelitis II.
CHARACTERISTICS OF PERITONEAL LYMPHOCYTES RESPONDING TO ANTIGEN
A. I. LEVINSON, R. P. LISAK, B. ZWEIMAN & L. D. WILKERSON Division of Allergy and Immunology, Department of Medicine and the Department of Neurology, School of Medicine, University of Pennsylvania; University of Pennsylvania and Wistar Institute Multiple Sclerosis Research Center, U.S.A.
Received 4 November 1976; acceptedfor publication 9 March 1977
Summary. Myelin basic protein (BP), induces an increased in vitro proliferative response of peritoneal exudate cells (PEC), compared with that given by peripheral blood lymphocytes (PBL), in guinea-pigs with experimental allergic encephalomyelitis (EAE). This response is determined by the nature of the lymphocytes in such exudates. We have also found that: (a) the majority of peritoneal lymphocytes from non-sensitized animals are E-rosetting (T cells) (59%) or null cells (>40%) with EAC-rosetting (B cells) comprising 1% or less of cells; (b) both T cells and null cells respond equally to BP as determined by a technique combining rosette-formation and autoradiography; (c) the increased in vitro peritoneal lymphocyte response in animals with EAE cannot be explained solely by the number of null cells since peripheral blood and lymph nodes also contain appreciable numbers of null cells.
to the sensitizing antigen than are comparable numbers of lymphoid cells from other compartments
(Rosenstreich, Blake & Rosenthal, 1971; Rosenthal, Rosenstreich, Davie & Blake, 1972). Studies from this laboratory have demonstrated an increased reactivity of peritoneal lymphocytes of animals with experimental allergic encephalomyelitis (EAE) to homologous myelin basic protein (GP-BP), the antigen that induces this experimental autoimmune disease (Lisak & Zweiman, 1973; Lisak, Zweiman, Kies & Driscoll, 1975; Lisak, Zweiman & Levinson, 1976). The reasons for the enhanced in vitro reactivity are not well-defined, since both peritoneal macrophages (Rosenstreich et al., 1971) and the lymphocytes found in peritoneal exudates (Rosenstreich et al., 1971; Lisak et al., 1976) could be responsible. In recent studies we concluded that it was unlikely that the greater reactivity of peritoneal lymphocytes than blood lymphocytes to GP-BP was attributable solely to the macrophages in the peritoneal exudate cell population (Lisak et al., 1976). It was conceivable that lymphocytes particularly responsive to GP-BP might contribute to the increased reactivity of the peritoneal exudate cell population. Accordingly, we have characterized the peritoneal lymphocytes that proliferate in vitro, identifying
INTRODUCTION
Peritoneal exudate inflammatory cell populations include lymphocytes that are more reactive in vitro Correspondence: Dr R. P. Lisak, Department of Neurology, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, U.S.A. B
621
622
A. I. Levinson et al.
T and B lymphocytes by resetting techniques in combination with autoradiographic assays of in vitro proliferation induced by GP-BP (Elfenbein, Shevach & Green, 1972).
MATERIALS AND METHODS
Immunological characteristics of cells Preparation of tissue lymphocyte suspensions. (a) Thymus and lymph node. Unsensitized male Hartley guinea-pigs, weighing 400-500 g were injected i.p. with sterile mineral oil (Marcol, Humble Oil, Houston, Texas) Three days later, these animals were killed and the cervical lymph nodes and thymuses were removed. After thorough washing in Hanks's balanced salt (HBSS, GIBCO, Grand Island, New York), single cell suspensions were prepared by teasing apart the tissues, pressing them through a 30-gauge stainless steel mesh screen, and excluding residual debris by gravity sedimentation. Cells were suspended at a concentration of 2 x 106/ml in HBSS. (b) Peritoneal exudate lymphocytes. Cells were obtained by peritoneal lavage 3 days after the i.p. injection of sterile mineral oil, washed twice with HBSS and incubated for 30 min on a glass wool, nylon wool column. The column was prepared by packing I 0 g of nylon wool (Fenwall Laboratories, Division of Travenol Labs, Inc., Morton Grove, Illinois) into a 10-ml vol. in the bottom of a 50-ml plastic syringe followed by glass wool (Fisher Scientific, King of Prussia, Pennsylvania) occupying a further 10-ml vol. Both materials were pre-washed with warmed HBSS containing 10 per cent foetal calf serum (FCS) v/v and the column was incubated at 370 prior to use. The cells eluted from the column consisted of 95-98%4 small- and medium-sized lymphocytes; the remainder were cells capable of phagocytozing oil and/or latex particles. The eluted cells were washed and resuspended in HBSS (10% FCS) at a concentration of 2 x 106 cells/ml. Cell viability was consistently greater than 98% judged by trypan blue exclusion. (c) Peripheral blood lymphocytes (PBL). Mononuclear cell suspensions were prepared from heparinized blood by centrifugation over FicollHypaque density gradients (Boyum, 1968). Cells at the interface consisted of 5-20 per cent monocytes (phagocytic mononuclear cells identified after incubation with 1 1 jpm latex particles) and lympho-
cytes. The cells were suspended at a concentration of 2 x 106 cells/ml. Determination of cell-surface characteristics. (a) E rosettes. 0-05 ml of a concentrated suspension of washed latex particles (I 1 pam diameter, Coulter Electronics, Hialeah, Florida) was added to duplicate tubes containing 0-2 ml of either thymus, lymph node, PBL or PEL suspensions. Following incubation for 45 min at 370 on a mechanical rotator the cells were washed and the supernatants replaced with 0-2 ml fresh HBSS. Equal volumes of a I % solution of freshly drawn rabbit red blood cells (RRBC) were added to the resuspended cells. Following incubation on a mechanical rotator for 15 min at 370 the cells were centrifuged at 200 g at 40 for 5 min and kept on ice for 1 h. Subsequently the pellets were gently resuspended and the numbers of rosettes were counted. Lymphocytes with three or more adherent RRBC were scored as rosettes. Cells containing at least two latex particles were considered to be monocyte/macrophages and were excluded from the counts. Two hundred mononuclear cells were counted routinely. (b) EAC rosettes. Equal volumes of lymphoid cells prepared as above, sheep red blood cells
(SRBC) sensitized with IgM haemolysin (Cordis Laboratories, Miami, Florida) and mouse complement (AKR serum, Jackson Laboratories, Bar Harbor, Maine) were incubated on a mechanical rotator for 30 min at 37°. The suspensions were then examined for the percentage of latex-negative cells which were rosette positive. Identification of BP-reactive lymphocytes in lymphocyte-enriched peritoneal exudates of EAE animals Animal sensitization. Male Hartley albino guineapigs (450-700 g) were injected i.d. over the sternum on day 0 with 100 ug of guinea-pig basic protein (GP-BP), prepared by the method of Diebler, Martenson & Kies (1972), in Freund's complete adjuvant (containing 100 ug M. tuberculosis H37R., Difco Laboratories, Detroit, Michigan, in 0-1 ml). This method of sensitization has consistently induced EAE in our laboratory (Lisak & Zweiman, 1974a; Lisak & Zweiman, 1974b; Lisak et al., 1975). On day 10, 30 ml of sterile mineral oil was injected i.p. On day 13, peritoneal cavities were lavaged and exudate cells were harvested as previously described (Lisak et al., 1974a; Lisak et al., 1976).
Reactive and non-reactive lymphocytes
Cell separation and culture techniques. Cells were washed twice in HBSS and passed over a column of pre-washed coiled cotton (Type A, Fisher Scientific) in order to enrich the lymphocyte population. The cells eluted from the column consisted of 50-80% small and medium lymphocytes, 5-10% polymorphonuclear cells, and 30-50% macrophages. The latter were identified by their ability to ingest oil and/or latex particles. These cotton filtered peritoneal exudate cells (PECcF) were suspended in Spinnermodified Eagle's Minimal Essential Medium (MEM-S, GIBCO) supplemented with penicillin, streptomycin, glutamine and FCS (l0%Yo v/v) and cultured for 4 days at a concentration of 1 x 106 lymphocytes/ml at 370 in 5% C02/air. To multiple replicate cultures were added either: (1) GP-BP in the final concentration of 25 ug/ml, or (2) additional culture medium. Following a terminal 18-h pulse of tritiated thymidine, the contents of three tubes of each replicate group were processed for the assay of isotope uptake by scintillation counting. The degree of proliferation was expressed as a stimulation index (SI) where: - mean
c.p.m. of GP-BP-stimulated cultures mean c.p.m. of control cultures
Rosetting-autoradiography assay. The remaining replicate cultures were assayed by a combination of rosetting and autoradiography. Replicate cultures were pooled; the cells were washed and E or EAC rosettes were formed. To preserve the integrity of the rosettes, glutaraldehyde in FCS (previously absorbed with RRBC) was added to the rosetting suspensions at a final concentration of 2-5% for E rosettes and 10% for EAC rosettes. Preliminary studies in our laboratory indicated that this modification did not significantly alter the percentage of rosettes as compared with our standard method. Rosetting suspensions were transferred to gelatin-coated glass slides and allowed to dry before final fixation in methanol. The slides were dipped in Kodak NTB-2 emulsion (Kodak, Rochester, New York) and incubated for 7 days at 40 for autoradiography as previously described (Phillips & Zweiman, 1970). After development and staining with Giemsa solution, at least 400 cells were examined for the percentages of E-rosetting lymphocytes, the percentage of labelled cells, the percentage of rosette cells that were labelled, and the percentage of labelled cells that formed rosettes. All cells that had
623
1ooL 80 Vn -
2 60 2 20
0
E EAC N
Thymus
E EAC N Lymph node
E EAC N
E EAC N
PBL
PELt
E EAC N PEC t
Figure 1. Lymphocyte sub-populations in non-sensitized guinea pigs. Mean ± s.e.m.; macrophage/monocytes excluded from the counts; * PBL =peripheral blood mononuclear cells; t PEL = peritoneal exudate lymphocytes; * PEC = unfiltered peritoneal exudate cells. E = E-rosetting (T) cells; EAC = EAC-rosetting (B) cells; N = null cells.
ingested at least two latex particles, presumably macrophages, were excluded from the counts.
RESULTS Surface characteristics of lymphocytes The prevalence of E-and EAC-rosetting cells in the PEL of unsensitized animals is indicated in Fig. 1. The majority (59%) of the PEL were T cells forming E rosettes; no EAC-rosetting cells were present. However, over 40% on average, of the PEL formed neither E nor EAC rosettes; hence, the term 'null cells' was applied. To rule out the possibility that these results were due to insensitive rosetting techniques, comparative studies of thymus, lymph node cells and PBL were made using the same assays. The large majority of thymocytes formed E rosettes (mean=92%) but not EAC rosettes. The mean percentages of E- and EACrosetting cells in the lymph node lymphocyte population were 55% and 31% respectively. The PBL populations contained a mean of 60% E and 13% EAC rosettes. These findings are similar to those reported elsewhere (Staedecker, Bishop & Wortis, 1973; Wilson & Coombs, 1973; Sandberg, Soder & Ernstrom, 1976) and attest to the sensitivity of the techniques. Thus the percentage of null cells was significantly higher in the normal PEL than in the thymus and lymph node cells, but not the PBL populations. To determine whether the cell enrichment
A. I. Levinson et al.
624
Table 1. Contributions of T and null cells to the proliferative response to GP-BP* in animals with EAE
Experiment
SIt
Per cent labelled cells
Per cent E-rosetting cells
1 2 3 4 5 6
8 26 16 17 50 6
71 40 26 33 43 9
45 35 23 31 45 18
Per cent labelled cells Per cent E rosette that form E rosettes cells that are labelled 37 26 27 27 50 48
60 29 23 28 37 23
* GP-BP = Guinea-pig basic protein. t SI=mean c.p.m. of GP-BP stimulated cultures mean c.p.m. of control cultures
procedure for obtaining PEL may have been responsible in part for the prominence of null cells in this population, additional rosetting studies were performed on unfiltered peritoneal exudate cells (PEC). These cells were incubated with latex beads before rosetting was attempted so that phagocytic cells could be excluded. As seen in Fig. 1, the percentage of lymphocytes which rosetted with neither E nor EAC preparations was higher in PEC than that found in PEL (mean percentage of null cells = 61%). This suggests that column filtration of the peritoneal exudate cells did not selectively enrich the null cell
population. In vitro proliferative response to BP We determined which cells within the peritoneal exudate cell population of animals with EAE proliferated in vitro in response to GP-BP. The column fractionation used to prepare PEL led to considerable reduction in cell yield (Rosenstreich & Rosenthal, 1974; Lisak et al., 1976), so that cell numbers were insufficient for the assays combining rosetting and autoradiography. The pooling of PEL from several inbred animals (Rosenstreich et al., 1971) was not feasible in the present experiments with outbred Hartley guinea-pigs. Therefore, an alternative method was used. Peritoneal exudate cells passed over coiled cotton columns (PECCF), were composed of 30-50% macrophages, 40-50% lymphocytes and 5-10% granulocytes. Of the lymphocytes, 10-40% were T lymphocytes and 55-70% were null cells. No EACrosetting cells were seen.
We have previously found that PECCF from EAE animals proliferated briskly when cultured with GP-BP as measured by scintillation spectrometry (Lisak et al., 1973; Lisak et al., 1976). In the present study PECcF cultured with and without GP-BP were assayed by a combination of rosetting and autoradiography and the results were compared. It can be seen (Table 1) that GP-BP induced the proliferation of both T cells and null cells. Fig. 2 illustrates an example of a labelled T (E rosetting) and a labelled non-E-rosetting cell (null cell). As expected, no EAC-rosetting cells were found in these cultures. The phagocytic cells remaining in the cultures were neither labelled nor rosette forming. The proliferative response measured by autoradiography was confirmed by concomitant scintillation counting studies (SI ranging from 6-50). In contrast, no significant proliferation was seen in unstimulated cultures (
