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Experimental allergic encephalomyelitis — Migration of early T cells from the circulation into the central nervous system
Journal of the Neurological Sciences, 1978, 36: 55-61 © Elsevier/North-Holland Biomedical Press
55
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS - - MIGRATION OF EARLY T CELLS FROM THE CIRCULATION INTO THE CENTRAL NERVOUS SYSTEM
UTE TRAUGOTT, SANFORD H. STONE and CEDRIC S. RAINE
Departments of Pathology (Neuropathology) and Neuroscience, The Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N. Y. 10461, and NIA1D, Bethesda, Md. 20014 (U.S.A.) (Received 31 August, 1977) (Accepted 6 October, 1977)
SUMMARY
Study of lymphocytes from the blood of guinea pigs with acute EAE induced by isologous spinal cord in adjuvant reconfirmed that in comparison to normals, the percentage of early (active or high affinity rosetting) T cells decreases dramatically and that these changes can be correlated with clinical signs. In addition, we have investigated matching samples of CNS infiltrating cells recovered by ultrasonication and have found that coinciding with the decrease in early T cells in the circulation, significantly higher levels (P < 0.001) of these cells appear within the CNS compartment. It is concluded that the decrease of early T cells in the circulation is caused by their migration to the target organ, the CNS.
INTRODUCTION
Although it is known that in experimental allergic encephalomyelitis (EAE) there is lack of correlation between morphologic changes in the central nervous system (CNS) and clinical signs (see Raine, Snyder, Stone and Bornstein 1977), a recent immunologic study has revealed marked fluctuations in certain lymphocyte populations which correlated well with the severity of clinical signs (Traugott and Raine 1977). This latter study revealed that in contrast to circulating B cell and late (total) T cell values, early (active or high affinity rosetting) T cell levels showed significant decreases which coincided with clinical worsenings in inbred Strain 13 guinea pigs sensitized for acute EAE. From this study (Traugott and Raine 1977), it was speculated that these decreases in circulating early T cells might be ascribed either to their selective destruction or to their migration from the blood, possibly to the CNS which is known to be invaded by mononuclear inflammatory cells. The present communication addresses
56 these alternatives by investigating the levels of early T cells dynamically in these twt~ compartments (blood and CNS) and has demonstrated that coinciding with decreased levels of early T cells in the blood, high percentages of these cells appeared within the meningeal infiltrates, probably reflecting migration from the circulation to the targel organ. MATERIAL AND METHODS
Normal animals Ten normal, adult (approximately 6 months of age) male, Strain 13 guinea pigs (Strain 13/N from NIH), were used. Body weights ranged from 450 to 650 g. Experimental animals Seven similar adult Strain 13 guinea pigs and 3 randomly bred Hartley strain guinea pigs were used. Body weights also ranged from 450 to 650 g. All animals were housed in groups of 2-4 per cage and fed cabbage, chow and water ad libitum. Induction of EAE Each guinea pig was inoculated intracutaneously in multiple sites in the nuchal area with 0.5 ml of an emulsion containing 0.25 ml of a 50 ~ suspension of isologous spinal cord in saline and 0.25 ml of complete Freund's adjuvant (CFA) containing 10 mg of killed M. tuberculosis/ml (Stone and Lerner 1965). Isolation of mononuclear cells from the blood Under anesthesia with ether, 3 ml of heparinized blood were obtained by cardiac puncture. Circulating mononuclear cells were isolated on a Ficoll-Hypaque gradient, as described previously (B6yum 1968; Traugott and Raine 1977). Isolation of inflammatory cells from the meninges Animals were anesthetized with ether and perfused with cold (4 °C) PBS to remove contaminating blood cells. After dissection, the meninges were separated from the spinal cord and both were ultrasonicated for 5-10 min at 4 °C in Medium 199, a procedure which resulted in a suspension of inflammatory cells released from the meninges and the surface of the spinal cord. The viability of the resultant cell preparation was about 70 ~, as tested by trypan blue exclusion. Lymphocyte surface markers T cells were determined by spontaneous, non-immune rosette formation with rabbit erythrocytes, described in detail previously (Wilson and Coombs 1973). Early (active) T cells which rosette promptly, were counted immediately after centrifugation and late (total) T cells, after incubation overnight at 4 °C. B cells were estimated by direct immunofluorescence using FITC-conjugated rabbit anti-guinea pig gammaglobulin (Rabellino, Cohen, Grey and Unanue 1971). It must be emphasized that the "B" cell population also includes Fc receptor bearing ceils (monoeytes) and the figure is probably highly inflated in the CNS preparations.
57
Statistical analysis Comparison between the lymphocyte levels in the blood of control and experimental animals was performed by applying the Student's t test. In addition, in experimental animals the differences between circulating lymphocyte populations and inflammatory cells rescued from the CNS were similarly analyzed. RESULTS
Normal guinea pigs (a) Circulating cells Heparinized blood samples were tested from 10 animals. The percentages of early T cells were 32.6 -+- 12.2~o (mean :~ 2 SD); for late T cells, 58.6 ~ 12.8 ~ ; and for B cells, 26.5 ± 8 . 2 ~ (see Table 1).
(b) Meningeal cells Lymphocyte percentages in the meninges of normal animals could not be determined because of the known low numbers of mononuclear cells normally present in this site.
EAE guinea pigs (a) Clinicalfindings As previously described (Stone and Lerner 1965), the first clinical signs were manifested between days 12 and 21 post-inoculation (PI). Animals became sick between days 12 and 21 PI and were sacrificed between days 13 and 24 PI (Fig. 1). From each of the 10 experimental animals, samples of blood and meningeal cells were taken and tested simultaneously. There were no differences in disease response or lymphocyte findings between the Strain 13 and Hartley strains.
(b) Lymphocyte values The results from the experimental animals (vide infra) were analyzed by comTABLE 1 LYMPHOCYTE VALUES IN BLOOD AND CNS INFILTRATES IN GUINEA PIGS
Early T cells Late T cells B cells
(A). Normal blood (mean ±2SD)(%)
Significance between(A) and(B)
(B). EAE blood (mean 4-2SD)(%)
Significance between(B) and(C)
32.6 ± 12.2 58.6 4- 12.8 26.5 :~ 8.2
P < 0.001 P < 0.001 P < 0.05
14.5 -4- 10.64 P < 0.001 44.5 i 25.9 not sig. 29.2 ± 12.9 P < 0.001
(C). EAE CNS cells (mean ±2SD)(%) 42.4 i 13.7 43.6 ± 14.9 34.8 dz 11.1a
This figure is highly inflated because in addition to B cells, it includes the higher levels of monocytes (34 ~) present in the CNS.
58 paring lymphocyte values from (i) the blood of experimental animals with that from normal animals; (ii) meningeal infiltrates from experimental animals with the blood of normal animals; and (iii) the blood and meningeal infiltrates from experimental animals. A comparison between meningeal cells from normal animals with other groups was not possible for the reason mentioned above. (i) Comparison of circulating cells from experimental and normal animals. The percentage of early T cells in experimental animals was 14.5 ~ 5- 10.64~o (mean :r 2 SD); of late T cells, 44.5 ~ ± 25.9 %; and of B cells, 29.2~ ± 12.9 yo (Table 1). Early T cells showed a significant decrease from normal values (32.6 ~ ± 12.2 %), ranging from 4 % to 22 Yo. These decreases correlated with the severity of clinical signs in that more severely affected animals showed lower percentages of early T cells. The decrease in circulating late T cells, found in 7 out of 10 animals, showed less correlation with the clinical picture. B cell levels showed minor changes only and did not correlate with clinical signs. These findings on circulating cells are in agreement with the results of our previous study (Traugott and Raine 1977).
(ii) Comparison of meningeal cells of EAE animals and blood cells of" normals. Meningeal cells were recovered from the heavily infiltrated CNS by ultrasonication in medium. The resultant cell suspension contained an abundance of mononuclear cells with little contamination by other elements. The meningeal infiltrates from EAE animals (Table 1) contained 42.4 4- 13.7% early T cells; 43.6 ± 14.9~o late T cells; and 34.8 ± 11.1 ~ "B" cells (see Methods). The mean value of early T cells from the CNS infiltrates (42.4 %) was higher than that found in the blood of normal guinea pigs (32.6%). Correlation between the percentages of early T cells in the meningeal cell suspensions and the severity of clinical signs was not detectable. In comparison to late T cell values in the blood of normal animals, the mean value of late T cells from the CNS of experimental animals was also decreased, whereas the percentages of B cells were slightly elevated. (iii) Comparison o] meningeal cells and circulating cells from EAE animals. The findings are presented in histogram form in Fig. 1. The most remarkable finding was a difference in early T cell percentages between the two compartments caused by the marked decrease in circulating early T cells and their high levels in the meninges. Statistically, this difference was highly significant (P < 0.00l). "B" cells were slightly elevated in the meningeal cells and late T cells showed no significant difference. DISCUSSION A recent immunologic study on EAE has revealed a strong correlation between early T cell populations in the blood and disease activity in that with increase in clinical severity, this population decreased dramatically (Traugott and Raine 1977). This implied that circulating early T cell levels were somehow related to the active disease process in the CNS. The present study has, for the first time, employed lymphocyte populations in inbred guinea pigs afflicted with acute EAE. Our findings have reconfirmed the low values of circulating early T cells coinciding with increasing severity of clinical signs and have shown high levels of this cell population occurring at the same
59
LYMPHOCYTESIN BLOODAND MENINGEALINFILTRATESIN ACUTEEAE I0 GUINEAPIGS - I0 SAMPLES SEVERITY OFCLINICALSIGNS t
t
t
iI~
11~
~
2
3
4
5
6
13
13
13
14
16
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~
II~
l
8
16
IT
9 18
60 F EARLY T CELLS
LATE T CELLS
80
-2S.D. 60 % 40 20 0 60,-
B CELLS
RANGE IN BLOOD J
G.p. i
DAYSPI 13
I0 24
Fig. 1. Actual values of circulating and CNS-associated lymphocytes from each of 10 acute EAE guinea pigs, shown in histogram form. In the normal CNS, the small number of mononuclear cells present is insufficient for the determination of lymphocyte subpopulations. Cross-hatched column = circulating lymphocytes, the adjacent empty column represents meningeal infiltrates, from the same animal. In the early T cell histogram (top), note the significantlyhigher levels of early T cells in the CNS, the decreased levels of circulating early T cells and the correspondence of the latter with the severity of clinical signs. In the late T cell histogram, note their unusual presence in the CNS, at values which are not significantly different from those in the circulation of EAE animals. Note the presence of "B" cells in the CNS at levels higher than those in the circulation of the same animals. At top, note clinical signs corresponding to the pair of columns from each animal: t = incontinence and/or slight paraparesis; tt : obvious paraparesis and/or ataxia; t t t = severe paraparesis --> quadriparesis. The appropriate range (mean 4- 2 SD) of lymphocytes from the circulation of normal guinea pigs is shown to the left in each histogram. Days PI : days post-inoculation. time within the C N S c o m p a r t m e n t . This s u p p o r t s o u r previous c o n t e n t i o n ( T r a u g o t t a n d R a i n e 1977) t h a t early T cells m i g h t be causally r e l a t e d to E A E a n d t h a t their d i m i n u t i o n f r o m the c i r c u l a t i o n m i g h t be explained by their m i g r a t i o n to the target organ, the C N S , r a t h e r t h a n by their selective d e s t r u c t i o n in the b l o o d . Statistical analysis o f early T cell levels in the circulation a n d the meningeal infiltrates in acute E A E has shown the differences to be h i g h l y significant ( P < 0.001), thus further indic a t i n g t h a t the fluctuations in the two c o m p a r t m e n t s were related. W i t h r e g a r d to
60 circulating B cells and late T cells in EAE and normal animals, these were examined i~ our previous report (Traugott and Raine 1977) and found to differ only slightly. From the present study, no difference was detected between late T cells from the blood and those from the meningeal infiltrates of EAE animals, while cells staining positively for immunofluorescence ("B" cells) were elevated in the CNS compartment. Whether the latter elevation represented a specific response cannot be claimed because within these meningeal infiltrates, a high percentage (34 ~) of monocytes was demonstrable, versus 5 ~o in the blood. Since these cells bear Fc receptors, they also stained positively by immunofluorescence and were included in the B cell values by the present technique. While a causative role for T cells in general in EAE is well established using peripheral lymphocytes (Paterson 1976), the involvement of early T cells in cellmediated immunity, in particular EAE, was discussed in our previous communication (Traugott and Raine 1977). The reasons underlying the appearance and accumulation of mononuclear cells within the CNS in EAE, in particularly early T cells, are not clearly understood since they could involve specific or non-specific migration or local proliferation. The paucity of mitotic figures and the inability to account for such large numbers of meningeal cells arising from the small number of pre-existing cells tend to argue against local proliferation being the main factor. That migration of mononuclear cells to the CNS is operative in EAE is likely and is indicated by the marked predilection for inflammation to occur within the CNS. The known affinity for lymphocytes to move actively to sites of inflammation regardless of antigen specificity (Asherson and Allwood 1972; McGregor and Logie 1974) probably indicates that many of the mononuclear cells in EAE infiltrates accumulate non-specifically. Thus, the question arises, what percentage of early T cells in the CNS demonstrates antigen specificity in the present system. To date, no direct evidence exists for high numbers of specific T cells in the CNS but recent work on circulating tymphocytes from guinea pigs with EAE by Hashim, Lee and Pierce (1977) suggests that a considerable percentage of active T cells is specifically sensitized to myelin basic protein (MBP) and that this population increases in the presence of MBP. Using different antigens, Felsburg and Edelman (1977) could not account for the elevation of active T cells being related to an increase in antigen specific cells. They proposed the increase to be due to the release of a soluble factor produced by the antigen-specific population after contact with the appropriate antigen. In some way, the surface receptors of non-specific lymphocytes were changed, thereby effecting an alteration in their affinity to form rosettes. This question has been raised previously several times. For example, using labelled lymphoid cells in passive transfers, Turk (1962) found equally small numbers of labelled cells in both specific and non-specific local skin test sites in recipients. Obviously in the present system, one would like to know what percentage of the early T cells displays MBP responsiveness, the subject of ongoing work in this laboratory. Since cell-mediated immunity, particularly to MBP, in multiple sclerosis (MS) the disease for which EAE serves as the prime experimental model - - is not fully established, it is difficult to extrapolate to the human disease from the findings presented here. Reports on circulating late (total) T cells in MS are conflicting (Lisak, Levinson, Zweiman and Abdou 1975; Oger, Arnason, Wray and Kistler 1975; Reddy and Goh -
-
61 1976) and no study on early T cells in MS has appeared as yet. However, an elevation of late (total) T cells in the cerebrospinal fluid, associated with exacerbations in MS, has been reported (Allen, Sheremata, Cosgrove, Osterland and Shea 1976). This might i m p l y a role for CNS-specific cellular i m m u n i t y in MS a n d m i g r a t i o n of these cells to the C N S d u r i n g active disease. F u t u r e work on lymphocyte p o p u l a t i o n s in MS should take the present observations on E A E into consideration. ACKNOWLEDGEMENTS
The authors thank Dr. Robert T. Terry for constructive criticism. Everett Swanson, Miriam Pakingan and Howard Finch are thanked for their expert technical assistance and Mary Palumbo and Violet Hantz for secretarial help. Supported in part by a grant from the National Multiple Sclerosis Society, R G 1001-A-1 ; by G r a n t s NS 08952 a n d NS 11920 from the U S P H S ; by a grant from the K r o c F o u n d a t i o n a n d a grant from the Alfred P. Sloan F o u n d a t i o n . D u r i n g this work, Dr. Raine was the recipient of a Research Career D e v e l o p m e n t A w a r d from the U S P H S NS 70265.
REFERENCES Allen, J. C., W. Sheremata, J. B. R. Cosgrove, U. Osterland and M. Shea (1976) Cerebrospinal fluid T and B lymphocyte kinetics related to exacerbations of multiple sclerosis, Neurology (Minneap.), 26: 579-583. Asherson, G. L. and G. G. Allwood (1972) Inflammatory lymphoid cells. Cells in immunised lymph nodes that move to sites of inflammation, Immunology, 22: 493-502. B6yum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood, Scand. J. clin. Lab. Invest., 21, Suppl. 97" 77-120. Felsburg, P. J. and R. Edelman (1977) The active E-rosette test: a sensitive in vitro correlate for human delayed-type hypersensitivity, J. lmmunol., 118: 62-66. Hashim, G. A., D. H. Lee and J. C. Pierce (1977) Antigen-stimulatedrosette formation by T lymphocytes in experimental allergic encephalomyelitis, Neurochem. Res., 2: 99-109. Lisak, R. P., A. 1. Levinson, B. Zweiman and N. I. Abdou (1975) T and B lymphocytes in multiple sclerosis, Clin. exp. Immunol., 22: 30-34. McGregor, D. D. and P. S. Logie (1974) The mediation of cellular immunity. VII (Localization of sensitized lymphocytes in inflammatory exudates), J. exp. Med., 139: 1415-1425. Oger, J. F., G. B. W. Arnason, S. H. Wray and J. P. Kistler (1975) A study of B and T cells in multiple sclerosis, Neurology (Minneap.), 25: 444-447. Paterson, P. Y. (1976) Experimental autoimmune (allergic) encephalomyelitis. Induction, pathogenesis and suppression. In: P. A. Meischer and H. J. Muller-Eberhard (Eds.), Textbook of lmmur, opathology, Grune and Stratton, New York, pp. 179-213. Rabellino, E., S. Cohen, H. M. Grey and E. R. Unanue (1971) Immunoglobulin on the surface of lymphocytes. I (Distribution and quantitation), J. exp. Med., 133:156-170. Raine, C. S., D. H. Snyder, S. H. Stone and M. B. Bornstein (1977) Suppression of acute and chronic experimental allergic encephalomyelitis in Strain 13 guinea pigs, J. neurol. Sei., 31 : 355-367. Reddy, M. M. and K. O. Goh (1976) B and T lymphocytes in man. III (B, T and "null" lymphocytes in multiple sclerosis), Neurology (Minneap.), 26: 997-999. Stone, S. H. and E. M. Lerner (1965) Chronic disseminated allergic encephalomyelitis in guinea pigs, Ann. N. Y. Acad. Sci,, 122: 227-241. Traugott, U. and C. S. Raine (1977) Experimental allergic encephalomyelitis in inbred guinea pigs. Correlation of decrease in early T cells with clinical signs in suppressed and unsuppressed animals, Cell. hnmunoL, 34: 146-155. Turk, J. L. (1962) The passive transfer of delayed hypersensitivity in guinea pigs by the transfusion of isotopically labelled lymphoid cells, Immunology, 5: 478488. Wilson, A. B. and R. R. A. Coombs (1973) Rosette formation between guinea pig lymphoid cells and rabbit erythrocytes - - a possible T cell marker, Int. Arch. Allergy, 44: 544-560.
55
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS - - MIGRATION OF EARLY T CELLS FROM THE CIRCULATION INTO THE CENTRAL NERVOUS SYSTEM
UTE TRAUGOTT, SANFORD H. STONE and CEDRIC S. RAINE
Departments of Pathology (Neuropathology) and Neuroscience, The Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N. Y. 10461, and NIA1D, Bethesda, Md. 20014 (U.S.A.) (Received 31 August, 1977) (Accepted 6 October, 1977)
SUMMARY
Study of lymphocytes from the blood of guinea pigs with acute EAE induced by isologous spinal cord in adjuvant reconfirmed that in comparison to normals, the percentage of early (active or high affinity rosetting) T cells decreases dramatically and that these changes can be correlated with clinical signs. In addition, we have investigated matching samples of CNS infiltrating cells recovered by ultrasonication and have found that coinciding with the decrease in early T cells in the circulation, significantly higher levels (P < 0.001) of these cells appear within the CNS compartment. It is concluded that the decrease of early T cells in the circulation is caused by their migration to the target organ, the CNS.
INTRODUCTION
Although it is known that in experimental allergic encephalomyelitis (EAE) there is lack of correlation between morphologic changes in the central nervous system (CNS) and clinical signs (see Raine, Snyder, Stone and Bornstein 1977), a recent immunologic study has revealed marked fluctuations in certain lymphocyte populations which correlated well with the severity of clinical signs (Traugott and Raine 1977). This latter study revealed that in contrast to circulating B cell and late (total) T cell values, early (active or high affinity rosetting) T cell levels showed significant decreases which coincided with clinical worsenings in inbred Strain 13 guinea pigs sensitized for acute EAE. From this study (Traugott and Raine 1977), it was speculated that these decreases in circulating early T cells might be ascribed either to their selective destruction or to their migration from the blood, possibly to the CNS which is known to be invaded by mononuclear inflammatory cells. The present communication addresses
56 these alternatives by investigating the levels of early T cells dynamically in these twt~ compartments (blood and CNS) and has demonstrated that coinciding with decreased levels of early T cells in the blood, high percentages of these cells appeared within the meningeal infiltrates, probably reflecting migration from the circulation to the targel organ. MATERIAL AND METHODS
Normal animals Ten normal, adult (approximately 6 months of age) male, Strain 13 guinea pigs (Strain 13/N from NIH), were used. Body weights ranged from 450 to 650 g. Experimental animals Seven similar adult Strain 13 guinea pigs and 3 randomly bred Hartley strain guinea pigs were used. Body weights also ranged from 450 to 650 g. All animals were housed in groups of 2-4 per cage and fed cabbage, chow and water ad libitum. Induction of EAE Each guinea pig was inoculated intracutaneously in multiple sites in the nuchal area with 0.5 ml of an emulsion containing 0.25 ml of a 50 ~ suspension of isologous spinal cord in saline and 0.25 ml of complete Freund's adjuvant (CFA) containing 10 mg of killed M. tuberculosis/ml (Stone and Lerner 1965). Isolation of mononuclear cells from the blood Under anesthesia with ether, 3 ml of heparinized blood were obtained by cardiac puncture. Circulating mononuclear cells were isolated on a Ficoll-Hypaque gradient, as described previously (B6yum 1968; Traugott and Raine 1977). Isolation of inflammatory cells from the meninges Animals were anesthetized with ether and perfused with cold (4 °C) PBS to remove contaminating blood cells. After dissection, the meninges were separated from the spinal cord and both were ultrasonicated for 5-10 min at 4 °C in Medium 199, a procedure which resulted in a suspension of inflammatory cells released from the meninges and the surface of the spinal cord. The viability of the resultant cell preparation was about 70 ~, as tested by trypan blue exclusion. Lymphocyte surface markers T cells were determined by spontaneous, non-immune rosette formation with rabbit erythrocytes, described in detail previously (Wilson and Coombs 1973). Early (active) T cells which rosette promptly, were counted immediately after centrifugation and late (total) T cells, after incubation overnight at 4 °C. B cells were estimated by direct immunofluorescence using FITC-conjugated rabbit anti-guinea pig gammaglobulin (Rabellino, Cohen, Grey and Unanue 1971). It must be emphasized that the "B" cell population also includes Fc receptor bearing ceils (monoeytes) and the figure is probably highly inflated in the CNS preparations.
57
Statistical analysis Comparison between the lymphocyte levels in the blood of control and experimental animals was performed by applying the Student's t test. In addition, in experimental animals the differences between circulating lymphocyte populations and inflammatory cells rescued from the CNS were similarly analyzed. RESULTS
Normal guinea pigs (a) Circulating cells Heparinized blood samples were tested from 10 animals. The percentages of early T cells were 32.6 -+- 12.2~o (mean :~ 2 SD); for late T cells, 58.6 ~ 12.8 ~ ; and for B cells, 26.5 ± 8 . 2 ~ (see Table 1).
(b) Meningeal cells Lymphocyte percentages in the meninges of normal animals could not be determined because of the known low numbers of mononuclear cells normally present in this site.
EAE guinea pigs (a) Clinicalfindings As previously described (Stone and Lerner 1965), the first clinical signs were manifested between days 12 and 21 post-inoculation (PI). Animals became sick between days 12 and 21 PI and were sacrificed between days 13 and 24 PI (Fig. 1). From each of the 10 experimental animals, samples of blood and meningeal cells were taken and tested simultaneously. There were no differences in disease response or lymphocyte findings between the Strain 13 and Hartley strains.
(b) Lymphocyte values The results from the experimental animals (vide infra) were analyzed by comTABLE 1 LYMPHOCYTE VALUES IN BLOOD AND CNS INFILTRATES IN GUINEA PIGS
Early T cells Late T cells B cells
(A). Normal blood (mean ±2SD)(%)
Significance between(A) and(B)
(B). EAE blood (mean 4-2SD)(%)
Significance between(B) and(C)
32.6 ± 12.2 58.6 4- 12.8 26.5 :~ 8.2
P < 0.001 P < 0.001 P < 0.05
14.5 -4- 10.64 P < 0.001 44.5 i 25.9 not sig. 29.2 ± 12.9 P < 0.001
(C). EAE CNS cells (mean ±2SD)(%) 42.4 i 13.7 43.6 ± 14.9 34.8 dz 11.1a
This figure is highly inflated because in addition to B cells, it includes the higher levels of monocytes (34 ~) present in the CNS.
58 paring lymphocyte values from (i) the blood of experimental animals with that from normal animals; (ii) meningeal infiltrates from experimental animals with the blood of normal animals; and (iii) the blood and meningeal infiltrates from experimental animals. A comparison between meningeal cells from normal animals with other groups was not possible for the reason mentioned above. (i) Comparison of circulating cells from experimental and normal animals. The percentage of early T cells in experimental animals was 14.5 ~ 5- 10.64~o (mean :r 2 SD); of late T cells, 44.5 ~ ± 25.9 %; and of B cells, 29.2~ ± 12.9 yo (Table 1). Early T cells showed a significant decrease from normal values (32.6 ~ ± 12.2 %), ranging from 4 % to 22 Yo. These decreases correlated with the severity of clinical signs in that more severely affected animals showed lower percentages of early T cells. The decrease in circulating late T cells, found in 7 out of 10 animals, showed less correlation with the clinical picture. B cell levels showed minor changes only and did not correlate with clinical signs. These findings on circulating cells are in agreement with the results of our previous study (Traugott and Raine 1977).
(ii) Comparison of meningeal cells of EAE animals and blood cells of" normals. Meningeal cells were recovered from the heavily infiltrated CNS by ultrasonication in medium. The resultant cell suspension contained an abundance of mononuclear cells with little contamination by other elements. The meningeal infiltrates from EAE animals (Table 1) contained 42.4 4- 13.7% early T cells; 43.6 ± 14.9~o late T cells; and 34.8 ± 11.1 ~ "B" cells (see Methods). The mean value of early T cells from the CNS infiltrates (42.4 %) was higher than that found in the blood of normal guinea pigs (32.6%). Correlation between the percentages of early T cells in the meningeal cell suspensions and the severity of clinical signs was not detectable. In comparison to late T cell values in the blood of normal animals, the mean value of late T cells from the CNS of experimental animals was also decreased, whereas the percentages of B cells were slightly elevated. (iii) Comparison o] meningeal cells and circulating cells from EAE animals. The findings are presented in histogram form in Fig. 1. The most remarkable finding was a difference in early T cell percentages between the two compartments caused by the marked decrease in circulating early T cells and their high levels in the meninges. Statistically, this difference was highly significant (P < 0.00l). "B" cells were slightly elevated in the meningeal cells and late T cells showed no significant difference. DISCUSSION A recent immunologic study on EAE has revealed a strong correlation between early T cell populations in the blood and disease activity in that with increase in clinical severity, this population decreased dramatically (Traugott and Raine 1977). This implied that circulating early T cell levels were somehow related to the active disease process in the CNS. The present study has, for the first time, employed lymphocyte populations in inbred guinea pigs afflicted with acute EAE. Our findings have reconfirmed the low values of circulating early T cells coinciding with increasing severity of clinical signs and have shown high levels of this cell population occurring at the same
59
LYMPHOCYTESIN BLOODAND MENINGEALINFILTRATESIN ACUTEEAE I0 GUINEAPIGS - I0 SAMPLES SEVERITY OFCLINICALSIGNS t
t
t
iI~
11~
~
2
3
4
5
6
13
13
13
14
16
11"
~
II~
l
8
16
IT
9 18
60 F EARLY T CELLS
LATE T CELLS
80
-2S.D. 60 % 40 20 0 60,-
B CELLS
RANGE IN BLOOD J
G.p. i
DAYSPI 13
I0 24
Fig. 1. Actual values of circulating and CNS-associated lymphocytes from each of 10 acute EAE guinea pigs, shown in histogram form. In the normal CNS, the small number of mononuclear cells present is insufficient for the determination of lymphocyte subpopulations. Cross-hatched column = circulating lymphocytes, the adjacent empty column represents meningeal infiltrates, from the same animal. In the early T cell histogram (top), note the significantlyhigher levels of early T cells in the CNS, the decreased levels of circulating early T cells and the correspondence of the latter with the severity of clinical signs. In the late T cell histogram, note their unusual presence in the CNS, at values which are not significantly different from those in the circulation of EAE animals. Note the presence of "B" cells in the CNS at levels higher than those in the circulation of the same animals. At top, note clinical signs corresponding to the pair of columns from each animal: t = incontinence and/or slight paraparesis; tt : obvious paraparesis and/or ataxia; t t t = severe paraparesis --> quadriparesis. The appropriate range (mean 4- 2 SD) of lymphocytes from the circulation of normal guinea pigs is shown to the left in each histogram. Days PI : days post-inoculation. time within the C N S c o m p a r t m e n t . This s u p p o r t s o u r previous c o n t e n t i o n ( T r a u g o t t a n d R a i n e 1977) t h a t early T cells m i g h t be causally r e l a t e d to E A E a n d t h a t their d i m i n u t i o n f r o m the c i r c u l a t i o n m i g h t be explained by their m i g r a t i o n to the target organ, the C N S , r a t h e r t h a n by their selective d e s t r u c t i o n in the b l o o d . Statistical analysis o f early T cell levels in the circulation a n d the meningeal infiltrates in acute E A E has shown the differences to be h i g h l y significant ( P < 0.001), thus further indic a t i n g t h a t the fluctuations in the two c o m p a r t m e n t s were related. W i t h r e g a r d to
60 circulating B cells and late T cells in EAE and normal animals, these were examined i~ our previous report (Traugott and Raine 1977) and found to differ only slightly. From the present study, no difference was detected between late T cells from the blood and those from the meningeal infiltrates of EAE animals, while cells staining positively for immunofluorescence ("B" cells) were elevated in the CNS compartment. Whether the latter elevation represented a specific response cannot be claimed because within these meningeal infiltrates, a high percentage (34 ~) of monocytes was demonstrable, versus 5 ~o in the blood. Since these cells bear Fc receptors, they also stained positively by immunofluorescence and were included in the B cell values by the present technique. While a causative role for T cells in general in EAE is well established using peripheral lymphocytes (Paterson 1976), the involvement of early T cells in cellmediated immunity, in particular EAE, was discussed in our previous communication (Traugott and Raine 1977). The reasons underlying the appearance and accumulation of mononuclear cells within the CNS in EAE, in particularly early T cells, are not clearly understood since they could involve specific or non-specific migration or local proliferation. The paucity of mitotic figures and the inability to account for such large numbers of meningeal cells arising from the small number of pre-existing cells tend to argue against local proliferation being the main factor. That migration of mononuclear cells to the CNS is operative in EAE is likely and is indicated by the marked predilection for inflammation to occur within the CNS. The known affinity for lymphocytes to move actively to sites of inflammation regardless of antigen specificity (Asherson and Allwood 1972; McGregor and Logie 1974) probably indicates that many of the mononuclear cells in EAE infiltrates accumulate non-specifically. Thus, the question arises, what percentage of early T cells in the CNS demonstrates antigen specificity in the present system. To date, no direct evidence exists for high numbers of specific T cells in the CNS but recent work on circulating tymphocytes from guinea pigs with EAE by Hashim, Lee and Pierce (1977) suggests that a considerable percentage of active T cells is specifically sensitized to myelin basic protein (MBP) and that this population increases in the presence of MBP. Using different antigens, Felsburg and Edelman (1977) could not account for the elevation of active T cells being related to an increase in antigen specific cells. They proposed the increase to be due to the release of a soluble factor produced by the antigen-specific population after contact with the appropriate antigen. In some way, the surface receptors of non-specific lymphocytes were changed, thereby effecting an alteration in their affinity to form rosettes. This question has been raised previously several times. For example, using labelled lymphoid cells in passive transfers, Turk (1962) found equally small numbers of labelled cells in both specific and non-specific local skin test sites in recipients. Obviously in the present system, one would like to know what percentage of the early T cells displays MBP responsiveness, the subject of ongoing work in this laboratory. Since cell-mediated immunity, particularly to MBP, in multiple sclerosis (MS) the disease for which EAE serves as the prime experimental model - - is not fully established, it is difficult to extrapolate to the human disease from the findings presented here. Reports on circulating late (total) T cells in MS are conflicting (Lisak, Levinson, Zweiman and Abdou 1975; Oger, Arnason, Wray and Kistler 1975; Reddy and Goh -
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61 1976) and no study on early T cells in MS has appeared as yet. However, an elevation of late (total) T cells in the cerebrospinal fluid, associated with exacerbations in MS, has been reported (Allen, Sheremata, Cosgrove, Osterland and Shea 1976). This might i m p l y a role for CNS-specific cellular i m m u n i t y in MS a n d m i g r a t i o n of these cells to the C N S d u r i n g active disease. F u t u r e work on lymphocyte p o p u l a t i o n s in MS should take the present observations on E A E into consideration. ACKNOWLEDGEMENTS
The authors thank Dr. Robert T. Terry for constructive criticism. Everett Swanson, Miriam Pakingan and Howard Finch are thanked for their expert technical assistance and Mary Palumbo and Violet Hantz for secretarial help. Supported in part by a grant from the National Multiple Sclerosis Society, R G 1001-A-1 ; by G r a n t s NS 08952 a n d NS 11920 from the U S P H S ; by a grant from the K r o c F o u n d a t i o n a n d a grant from the Alfred P. Sloan F o u n d a t i o n . D u r i n g this work, Dr. Raine was the recipient of a Research Career D e v e l o p m e n t A w a r d from the U S P H S NS 70265.
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