Clin. exp. Immunol. (1978) 34, 87-91.
Cerebrospinal fluid lymphocytes in experimental allergic encephalomyelitis L. D. W ILKERSON, R. P. L ISAK & B. ZWEIMAN Departments of Neurology and Pediatrics and the Allergy and Immunology Section, Department ofMedicine, University ofPennsylvania School ofMedicine and the University of PennsylvaniaWistar Institute Multiple Sclerosis Research Center, Philadelphia, Pennsylvania, USA
(Received 19 April 1978) SUMMARY
We report characteristics of the cerebrospinal fluid (CSF) pleocytosis (616±148 cells/jil) that occurred in guinea-pigs with definite clinical experimental allergic encephalomyelitis developing 12 to 16 days after sensitization with homologous myelin basic protein. This pleocytosis was not present in the cerebrospinal fluid of a group of animals studied when still healthy, 9 or 10 days after similar sensitization. Eighty-nine per cent of cells in the CSF pleocytosis were small lymphocytes, 8% were larger lymphocytes and the remainder mostly monocytes. Of the lymphocytes, most were E-rosetting or null cells. B-cell markers were uncommon. The cellular patterns in this CSF pleocytosis appear to be similar to those seen in some delayed hypersensitivity responses. INTRODUCTION Pathological and immunological features of experimental allergic encephalomyelitis (EAE) are often likened to those of delayed hypersensitivity responses (Alvord, 1970). Evidence that cell-mediated immunity is a major pathogenetic mechanism in this disease model has come from in vitro and passive transfer studies utilizing lymphoid cells from blood, lymph node or peritoneal exudate, but not cells actually comprising the lesion. The difficulty of isolating inflammatory cells dispersed into the tissue of the nervous system has limited the in vitro characterization of the inflammatory cells actually participating in the lesion. However, the pathological observation of a leptomeningeal inflammatory infiltrate in at least some animals with EAE (Waksman & Adams, 1962) suggests that a pleocytosis might occur in CSF, a pool of cells more accessible for characterization. Here we report documentation of a CSF pleocytosis and the preliminary characterization of the cells comprising it. We chose the guinea-pig, despite its small size, because well established parameters of cell-mediated immunity to basic protein and other antigens may be assessed in this species (Lisak & Zweiman, 1974a).
MATERIALS AND METHODS Animal sensitization. EAE was induced in 450-500 gm male albino Hartley guinea-pigs by intradermal injection of 100 fig homologous myelin basic protein, prepared by the method of Diebler, Martenson & Kies (1972) in Freund's complete adjuvant containing 100 fig mycobacterium H37Ra (Difco Laboratories, Detroit, Michigan) in the shaved sternal area. In earlier studies in our laboratory (Lisak & Zweiman, 1974a,b; Levinson et al., 1977) this method has reliably produced disease in most such outbred animals, usually with onset 12 to 18 days after sensitization. Animals were weighed and examined daily. One group was followed until definite clinical signs of EAE developed, consisting of weight loss associated with definite hind limb paralysis, sphincteric incontinence and altered righting response. For this group, studies were carried out as soon as clear-cut neurological signs of EAE appeared, ranging from 12 to 16 days after sensitization. A second group was studied 9 or 10 days after similar sensitization. This point was chosen because with this model we have rarely observed pathological Correspondence: Dr L. D. Wilkerson, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA. 0099-9104/78/0100-0087$02.00 (D 1978 Blackwell Scientific Publications
87
88
L. D. Wilkerson, R. P. Lisak & B. Zweiman
lesions and never seen clinical manifestations of EAE at that point, yet there is evidence of in vitro responsiveness to the sensitizing antigens (Lisak et al., 1974a). All the animals in this second group were clinically healthy. A third group consisted ofunsensitized normal controls. Cerebrospinalfluid (CSF). CSF was obtained from animals anaesthetized with tribomoethanol/amyl alcohol by puncture of the cervico-occipital membrane reached through mid-line dissection. Puncture with a 27 gauge needle under direct visual control allowed CSF to be retrieved by micropipette with minimal contamination by the peripheral blood. Cytology. CSF cells were fixed in gluteraldehyde (3 75%, pH 7 4), collected on to a membrane (Metricel, GAI, Gellman Instruments, Ann Arbor, Michigan), and placed in a gentle vacuum manifold. The membrane was stained by a modified Papanicolaou technique, cleared to translucency in xylene and mounted in Eukit. A specially designed centering device permitted convenient detection of at least 200 cells for inspection of cytological characteristics and measurement of cell diameter with an optical micrometer. Cytological identification of cells in CSF, including cells of nervous system origin, was made using the criteria of Kolmel (1976) Cell surface characteristics. Heparinized peripheral blood was fractionated by the method of Boyum (1968) to secure the mononuclear-rich layer. Mononuclear cells suspensions (5 x 105) were washed three times and studied for rosette formation with rabbit erythrocytes (E-rosettes) using a modification of the technique of Staedecker, Bishop & Wortis (1975) as previously detailed (Levinson et al., 1977). Using similar sized aliquots, rosette formation with sheep erythrocytes coated with antibody and complement (EAC-rosettes) was studied by the method described previously (Levinson et al., 1977). The small number of CSF cells available for study necessitated miniaturization of the techniques for rosette formation. This was first adapted with the peripheral mononuclear suspension and the results of the micromethod compared to those obtained by standard techniques applied to the same sample. For the micromethod, aliquots of 7500 cells each were suspended in 100 pl gelatin veronal buffer (GVB) containing 10% (v/v) foetal calf serum (FCS), and mixed with an equal volume of either 0-2% rabbit erythrocyte suspension (E-rosetting suspension) or 0-2% suspension of sheep erythrocyte coated with antibody and complement (EAC-rosetting suspension). EAC-rosettes were counted immediately following 30 min incubation with slow rotation of 370C. E-rosette preparations were similarly incubated for 15 min, centrifuged for 5 min at 200k at 4VC, incubated 60 min at 0C and then gently resuspended. Both resetting suspensions contained latex particles, and cells containing these were excluded from calculations of lymphocytes. Identification of mononuclear cells was facilitated by use of acridine orange or methylene blue, a particularly helpful step in E-rosetting given the similarity in size of guinea-pig mononuclear cells and rabbit erythrocytes. The same respective micromethods of E- and EAC-rosette formation were applied to aliquots of CSF containing 7500 leucocytes each. To maximize the yield of CSF cells available for rosette formation, washing was omitted and the unfractionated CSF aliquot was diluted to 100 J1d with GVB containing FCS (10% final v/v). For each CSF sample studied, the corresponding peripheral blood sample was studied by both micro and standard methods of rosette formation.
RESULTS With the technique as described above, CSF samples of about 100 jl were usually obtained. Only samples of CSF containing fewer than 5000 red blood cells (RBC) per tl were studied. In such specimens fewer than five leucocytes per pl in the CSF sample could be attributed to traumatic contamination. The degree of minimal RBC contamination was similar in specimens from normals (CSF RBC/pll = 2431 ± 513), specimens obtained from animals 9 or 10 days after sensitization (1427±535) and those obtained from animals with clinical manifestations ofEAE (2478±517). Table 1 lists the CSF leucocyte counts from respective groups of normals, of animals still healthy TABLE 1. CSF white cell counts
(B)
(C) Clinical EAE
(cells/pl)
Day 9/10 (cells/! i)
8 9-40 20 6+3-75
6 0-27 12-8+3-75
7 126-1588
(A)
Normals n Range Mean ± s.e.m.
(cells/pl)
616+148*
* Greater than (A) or (B) at P< 0 005. CSF white cells/jl in normal guinea-pigs (A); animals still healthy between 9 or 10 days after sensitization (B); and animals with signs ofdefinite EAE 12 to 16 days after sensitization (C).
CSF lymphocytes in EAE
89
9 or 10 days after sensitization, and of animals with clinically certain EAE. Small numbers of cells, 9-40/gl, were found in the CSF of normals. Nearly all these cells appeared to be small lymphocytes. Occasional monocytes were seen but arrachnoid, ependymal or polymorphonuclear leucocytes were not found. Similar total (0-27/gl) and differential cell counts were seen in CSF of animals studied 9 or 10 days after sensitization. In comparison, the CSF leucocyte counts from animals with clinical EAE (126-1588/gl) were significantly greater than those ofthe other groups (P< 0 005 in each case). Morphologically, most (88 4±2 2%) cells in the CSF of animals with frank EAE were small lymphocytes. Larger lymphocytes with more abundant cytoplasm and less dense nuclear staining were less frequent (8.3 ±21%). Size alone, as measured by an optical micrometer, was not the paramount distinguishing feature as occasional cells with larger cell diameters lacked other juvenile features. Small numbers of monocytes (range 1-4%), polymorphonuclear leucocytes (range 0-3%) and rare arrachnoid, ependymal or neural cells (range 0-0-5%) were seen. The portion of cells containing latex particles (3-0±1-3%) was similar to that having monocyte morphology. Table 2 shows the comparison of results of E- and EAC-rosetting by standard and micromethods on matching aliquots of peripheral mononuclear cell suspensions. With the standard technique, 400 cells of TABLE 2. Comparison of standard and micromethod rosetting techniques in corresponding PBL samples
Mean Greatest
E-Rosettes (%) Standard* Micromethodt
EAC-Rosettes (%) Standard Micromethod
62-6±1-8
8-4±2-5
discrepancy (%)
60-3±3-0
4-1±1-7
10
8
n = 7. *
Utilizing aliquots of 5 x 105 cells. t Utilizing aliquots of7500 cells.
the original 5 x 105 mononuclear cells were inspected, while with the micromethod 200 cells of the x 103 cells used were inspected. Although slightly lower values were obtained by micromethod, the paired results obtained by the two methods appeared to be in reasonable agreement. The lymphocyte sub-populations in the CSF of animals with clinically certain EAE are shown in Table 3. The majority of these lymphocytes were E-rosetting (59.3-44%). Comparison of the relative
7.5
TABLE 3. Lymphocyte subpopulation in CSF of animals with EAE
E-Rosettes (%) CSF Blood*
61 48 70 69 40 69 58 Mean s.e.m.
59.3 4-4
56 52 5 49.5 69 67 69-5 61-5 60-9 2-9
EAC-Rosette (%) CSF Blood* 1 0 5 0 3 1
Null Cells (%) CSF Blood
5 10 5 19 5 1
39 53 25 31 57 30
7-6t
39-2
n.t.
1-7 0-82
39 52 36 12 29 30 n.t.
2-6
5.4
33-0 5.4
* Percentage of rosettes as determined by micromethod applied to corresponding samples of CSF and PBL. t Blood greater than CSF P< 0 1 (paired t-test analysis). n.t. = Not tested.
90
L. D. Wilkerson, R. P. Lisak & B. Zweiman
lymphocyte sub-populations in blood and CSF of individual animals showed poor correlation (r = 0. 16) even for the E-rosetting cells, despite their predominance in both compartments. The discrepancy between findings in CSF and blood was striking in terms of EAC-rosettes. They were infrequent or not identified in some samples of CSF despite reasonable prevalence in the corresponding blood sample. Because few EAC-rosetting lymphocytes were found in CSF, even when as many as 19% of peripheral blood mononuclear cells were of this type, additional studies were carried out to ascertain whether certain factors may have led to falsely low CSF levels. First, all available CSF lymphocytes (40,000 and 100,000) from two animals with EAE were used to determine EAC-rosette formation, and the results were still within the group variability. Secondly, aliquots of 7500 peripheral blood mononuclear cells from each of two normals were suspended in cell-free supernatant of CSF from two animals with EAE and incubated for 1 hr at 370C. The results of EAC-rosetting of mononuclear cells suspended in supernatant of CSF were compared to the results of micro and standard methods of EAC-rosette formation applied to other aliquots of the respective sample suspended in GVB. EAC-rosetting in the supernatant of CSF of animals with EAE gave results (6% and 1%) similar to those for matching aliquots rosetted in GVB (10% and 2%, respectively). DISCUSSION In 1951, Kabat et al. documented an increase in the proportion of gammaglobulin in the CSF protein of monkeys with EAE and noted, as had Rivers & Schwentker (1935), that samples of CSF from some, but not all, of the sick animals contained white cells. With this exception, we are not aware of prior notation of CSF pleocytosis during EAE, though occurrence of such a pleocytosis would be predictable from the findings of Waksman & Adams (1962) that a leptomeningeal round cell infiltrate was a prominent early pathological feature in most of their guinea-pigs. In the present study a moderate pleocytosis in CSF accompanied EAE in all neurologically sick animals. It was not found in CSF of any ofthe second group of animals studied at a time (9 or 10 days after sensitization) when previous work had shown that similarly injected animals manifested in vivo or in vitro evidence of sensitivity to the inducing antigen, although they still appeared healthy (Lisak & Zweiman, 1974a). The finding that virtually all cells in the pleocytosis were mononuclear parallels the absence of polymorphonuclear leucocytes and other cells from the round cell leptomeningeal infiltrate described by Waksman & Adams (1962). While the source of these cells found in the CSF of animals with EAE has yet to be defined, several potential means of accumulation can be discounted. First, these findings are unlikely to have been simply an artefact produced by the minimal contamination of the CSF by bleeding. Extrapolation from the ratios of RBC in CSF and the blood of individual animals suggests that only a very small number of leucocytes in CSF specimens could be explained by blood contamination. The difference between the proportion of lymphocytes found in CSF (mean 96.6%) and in blood (mean 27.6%) also discounts the likelihood that the pleocytosis observed should be attributed to an artefact of traumatic sampling. Further, the characteristics of the pleocytosis suggest that it is likewise not attributable to a diffuse or unselective alteration of the blood: CSF barrier. The lack of correlation between CSF and peripheral blood leucocyte counts, and the marked differences in differential counts in the respective compartments suggest a more selective process of cell accumulation. Although lymphocytes in both compartments were predominantly T-cells or null cells, there was a poor correlation between the proportions of T-cells and null cells in CSF and the blood of individual animals and a striking paucity of identified B-cells and monocytes in CSF. These differences are also more consistent with a process of selective cell accumulation. The constituency of the CSF pleocytosis in the animals with EAE is broadly consistent with that of a delayed hypersensitivity response although exactly what constitutes the latter varies with the animal species and with different models within the species, as emphasized by McCluskey & Leter (1974). Preliminary characterization of this pleocytosis suggests a parallel with the earlier observation of Waksman & Adams (1962) that most mononuclear cells in early lesions of EAE appeared to be lymphocytes. Whether the mononuclear cell subpopulations in CSF and within the parenchyma are parallel remains
CSF lymphocytes in EAE
91
to be shown. For the present, these observations suggest a compartmentalization of cells between CSF and blood during the course of a presumed delayed hypersensitivity response to a nervous system constituent, myelin basic protein. The question raised is whether and how such a compartmentalization might be related to the mounting ofthe hypersensitivity response. The superb help of Ms. Felicula Mercado is happily acknowledged. Dr P. Doherty taught us cisternal puncture methods which we adapted for this study. This study was supported by Grant No. 5 P20 NS11037-10 and the National Multiple Sclerosis Society Grant No. 894-B-2. REFERENCES
peritoneal lymphocytes responding to antigen. ImmuALVORD, E.C. JR. (1970) Acute disseminated encephalonology. 33, 621. myelitis and 'allergic' neuroencephalopathies, in multiple sclerosis and other demyelinating diseases. Handbook of LIsAm, R.P. & ZWEIMAN, B. (1974a) In vitro and in vivo responses to homologous myelin basic protein in experiClinical Neurology (ed. by P. J. Vinken and G. W. Bruyn), mental allergic encephalomyelitis. Cell. Immunol. 11, 212. Vol. 9, p. 500. North-Holland, Amsterdam. BOYUM, A. (1968) Isolation of mononuclear cells and LISAK, R.P. & ZWEIMAN, B. (1974b) Immune responses to myelin basic protein in mycobacterial induced suppression granulocytes from human blood: Isolation of monoof experimental allergic encephalomyelitis. Cell. Immunol. nuclear cells by one centrifugation and granulocytes by combining centrifugation and sedimentation at 1 g. 14,242. MCCLUSKEY, R.T. & LETER, P.D. (1974) Cell-mediated Scand.j. Lab. Invest. 21, Suppl. 97, 77. reactions in vivo. Mechanisms in Cell-mediated Immunity DIEBLER, G.E., MARTENSON, R.E. & KIES, M.W. (1972) (ed. by R.T. McCluskey and S. Cohen), p. 1. Wiley, Large scale preparation of myelin basic protein from nervous system tissue of several mammalian species. New York. Prep. Biochem. 2, 139. RIVERS, T.M. & SCHWENTKER, F.F. (1935) EncephalomyeKABAT, E.A., WOLF, A., BEZER, A.E. & MURRAY, J.P. (1951) litis accompanied by myelin destruction experimentally Studies on acute disseminated encephalomyelitis proproduced in monkeys. J. exp. Med. 61, 689. duced experimentally in rhesus monkeys. VI. Changes in STAEDECKER, M.J., BISHOP, G. & WORTIS, H.H. (1973) cerebrospinal fluid proteins. _. exp. Med. 93, 615. Rosette formation by guinea pig thymocytes and thymus KOLMEL, H.W. (1976) Atlas of Cerebrospinal Fluid Cells. derived lymphocytes with rabbit red blood cells. J. Springer-Verlag, New York. Immunol. 11, 1834. LEVINSON, A., LisAK, R.P., ZWEIMAN, B. & WILKERSON, L.D. WAKSMAN, B.H. & ADAMS, R.D. (1962) A histologic study (1977) Reactive and non-reactive lymphocytes in experiof the early lesions in experimental allergic encephalomyemental allergic encephalomyelitis. II. Characteristics of litis in the guinea pig and rabbit. Amer. J. Path. 41, 135.
Cerebrospinal fluid lymphocytes in experimental allergic encephalomyelitis L. D. W ILKERSON, R. P. L ISAK & B. ZWEIMAN Departments of Neurology and Pediatrics and the Allergy and Immunology Section, Department ofMedicine, University ofPennsylvania School ofMedicine and the University of PennsylvaniaWistar Institute Multiple Sclerosis Research Center, Philadelphia, Pennsylvania, USA
(Received 19 April 1978) SUMMARY
We report characteristics of the cerebrospinal fluid (CSF) pleocytosis (616±148 cells/jil) that occurred in guinea-pigs with definite clinical experimental allergic encephalomyelitis developing 12 to 16 days after sensitization with homologous myelin basic protein. This pleocytosis was not present in the cerebrospinal fluid of a group of animals studied when still healthy, 9 or 10 days after similar sensitization. Eighty-nine per cent of cells in the CSF pleocytosis were small lymphocytes, 8% were larger lymphocytes and the remainder mostly monocytes. Of the lymphocytes, most were E-rosetting or null cells. B-cell markers were uncommon. The cellular patterns in this CSF pleocytosis appear to be similar to those seen in some delayed hypersensitivity responses. INTRODUCTION Pathological and immunological features of experimental allergic encephalomyelitis (EAE) are often likened to those of delayed hypersensitivity responses (Alvord, 1970). Evidence that cell-mediated immunity is a major pathogenetic mechanism in this disease model has come from in vitro and passive transfer studies utilizing lymphoid cells from blood, lymph node or peritoneal exudate, but not cells actually comprising the lesion. The difficulty of isolating inflammatory cells dispersed into the tissue of the nervous system has limited the in vitro characterization of the inflammatory cells actually participating in the lesion. However, the pathological observation of a leptomeningeal inflammatory infiltrate in at least some animals with EAE (Waksman & Adams, 1962) suggests that a pleocytosis might occur in CSF, a pool of cells more accessible for characterization. Here we report documentation of a CSF pleocytosis and the preliminary characterization of the cells comprising it. We chose the guinea-pig, despite its small size, because well established parameters of cell-mediated immunity to basic protein and other antigens may be assessed in this species (Lisak & Zweiman, 1974a).
MATERIALS AND METHODS Animal sensitization. EAE was induced in 450-500 gm male albino Hartley guinea-pigs by intradermal injection of 100 fig homologous myelin basic protein, prepared by the method of Diebler, Martenson & Kies (1972) in Freund's complete adjuvant containing 100 fig mycobacterium H37Ra (Difco Laboratories, Detroit, Michigan) in the shaved sternal area. In earlier studies in our laboratory (Lisak & Zweiman, 1974a,b; Levinson et al., 1977) this method has reliably produced disease in most such outbred animals, usually with onset 12 to 18 days after sensitization. Animals were weighed and examined daily. One group was followed until definite clinical signs of EAE developed, consisting of weight loss associated with definite hind limb paralysis, sphincteric incontinence and altered righting response. For this group, studies were carried out as soon as clear-cut neurological signs of EAE appeared, ranging from 12 to 16 days after sensitization. A second group was studied 9 or 10 days after similar sensitization. This point was chosen because with this model we have rarely observed pathological Correspondence: Dr L. D. Wilkerson, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA. 0099-9104/78/0100-0087$02.00 (D 1978 Blackwell Scientific Publications
87
88
L. D. Wilkerson, R. P. Lisak & B. Zweiman
lesions and never seen clinical manifestations of EAE at that point, yet there is evidence of in vitro responsiveness to the sensitizing antigens (Lisak et al., 1974a). All the animals in this second group were clinically healthy. A third group consisted ofunsensitized normal controls. Cerebrospinalfluid (CSF). CSF was obtained from animals anaesthetized with tribomoethanol/amyl alcohol by puncture of the cervico-occipital membrane reached through mid-line dissection. Puncture with a 27 gauge needle under direct visual control allowed CSF to be retrieved by micropipette with minimal contamination by the peripheral blood. Cytology. CSF cells were fixed in gluteraldehyde (3 75%, pH 7 4), collected on to a membrane (Metricel, GAI, Gellman Instruments, Ann Arbor, Michigan), and placed in a gentle vacuum manifold. The membrane was stained by a modified Papanicolaou technique, cleared to translucency in xylene and mounted in Eukit. A specially designed centering device permitted convenient detection of at least 200 cells for inspection of cytological characteristics and measurement of cell diameter with an optical micrometer. Cytological identification of cells in CSF, including cells of nervous system origin, was made using the criteria of Kolmel (1976) Cell surface characteristics. Heparinized peripheral blood was fractionated by the method of Boyum (1968) to secure the mononuclear-rich layer. Mononuclear cells suspensions (5 x 105) were washed three times and studied for rosette formation with rabbit erythrocytes (E-rosettes) using a modification of the technique of Staedecker, Bishop & Wortis (1975) as previously detailed (Levinson et al., 1977). Using similar sized aliquots, rosette formation with sheep erythrocytes coated with antibody and complement (EAC-rosettes) was studied by the method described previously (Levinson et al., 1977). The small number of CSF cells available for study necessitated miniaturization of the techniques for rosette formation. This was first adapted with the peripheral mononuclear suspension and the results of the micromethod compared to those obtained by standard techniques applied to the same sample. For the micromethod, aliquots of 7500 cells each were suspended in 100 pl gelatin veronal buffer (GVB) containing 10% (v/v) foetal calf serum (FCS), and mixed with an equal volume of either 0-2% rabbit erythrocyte suspension (E-rosetting suspension) or 0-2% suspension of sheep erythrocyte coated with antibody and complement (EAC-rosetting suspension). EAC-rosettes were counted immediately following 30 min incubation with slow rotation of 370C. E-rosette preparations were similarly incubated for 15 min, centrifuged for 5 min at 200k at 4VC, incubated 60 min at 0C and then gently resuspended. Both resetting suspensions contained latex particles, and cells containing these were excluded from calculations of lymphocytes. Identification of mononuclear cells was facilitated by use of acridine orange or methylene blue, a particularly helpful step in E-rosetting given the similarity in size of guinea-pig mononuclear cells and rabbit erythrocytes. The same respective micromethods of E- and EAC-rosette formation were applied to aliquots of CSF containing 7500 leucocytes each. To maximize the yield of CSF cells available for rosette formation, washing was omitted and the unfractionated CSF aliquot was diluted to 100 J1d with GVB containing FCS (10% final v/v). For each CSF sample studied, the corresponding peripheral blood sample was studied by both micro and standard methods of rosette formation.
RESULTS With the technique as described above, CSF samples of about 100 jl were usually obtained. Only samples of CSF containing fewer than 5000 red blood cells (RBC) per tl were studied. In such specimens fewer than five leucocytes per pl in the CSF sample could be attributed to traumatic contamination. The degree of minimal RBC contamination was similar in specimens from normals (CSF RBC/pll = 2431 ± 513), specimens obtained from animals 9 or 10 days after sensitization (1427±535) and those obtained from animals with clinical manifestations ofEAE (2478±517). Table 1 lists the CSF leucocyte counts from respective groups of normals, of animals still healthy TABLE 1. CSF white cell counts
(B)
(C) Clinical EAE
(cells/pl)
Day 9/10 (cells/! i)
8 9-40 20 6+3-75
6 0-27 12-8+3-75
7 126-1588
(A)
Normals n Range Mean ± s.e.m.
(cells/pl)
616+148*
* Greater than (A) or (B) at P< 0 005. CSF white cells/jl in normal guinea-pigs (A); animals still healthy between 9 or 10 days after sensitization (B); and animals with signs ofdefinite EAE 12 to 16 days after sensitization (C).
CSF lymphocytes in EAE
89
9 or 10 days after sensitization, and of animals with clinically certain EAE. Small numbers of cells, 9-40/gl, were found in the CSF of normals. Nearly all these cells appeared to be small lymphocytes. Occasional monocytes were seen but arrachnoid, ependymal or polymorphonuclear leucocytes were not found. Similar total (0-27/gl) and differential cell counts were seen in CSF of animals studied 9 or 10 days after sensitization. In comparison, the CSF leucocyte counts from animals with clinical EAE (126-1588/gl) were significantly greater than those ofthe other groups (P< 0 005 in each case). Morphologically, most (88 4±2 2%) cells in the CSF of animals with frank EAE were small lymphocytes. Larger lymphocytes with more abundant cytoplasm and less dense nuclear staining were less frequent (8.3 ±21%). Size alone, as measured by an optical micrometer, was not the paramount distinguishing feature as occasional cells with larger cell diameters lacked other juvenile features. Small numbers of monocytes (range 1-4%), polymorphonuclear leucocytes (range 0-3%) and rare arrachnoid, ependymal or neural cells (range 0-0-5%) were seen. The portion of cells containing latex particles (3-0±1-3%) was similar to that having monocyte morphology. Table 2 shows the comparison of results of E- and EAC-rosetting by standard and micromethods on matching aliquots of peripheral mononuclear cell suspensions. With the standard technique, 400 cells of TABLE 2. Comparison of standard and micromethod rosetting techniques in corresponding PBL samples
Mean Greatest
E-Rosettes (%) Standard* Micromethodt
EAC-Rosettes (%) Standard Micromethod
62-6±1-8
8-4±2-5
discrepancy (%)
60-3±3-0
4-1±1-7
10
8
n = 7. *
Utilizing aliquots of 5 x 105 cells. t Utilizing aliquots of7500 cells.
the original 5 x 105 mononuclear cells were inspected, while with the micromethod 200 cells of the x 103 cells used were inspected. Although slightly lower values were obtained by micromethod, the paired results obtained by the two methods appeared to be in reasonable agreement. The lymphocyte sub-populations in the CSF of animals with clinically certain EAE are shown in Table 3. The majority of these lymphocytes were E-rosetting (59.3-44%). Comparison of the relative
7.5
TABLE 3. Lymphocyte subpopulation in CSF of animals with EAE
E-Rosettes (%) CSF Blood*
61 48 70 69 40 69 58 Mean s.e.m.
59.3 4-4
56 52 5 49.5 69 67 69-5 61-5 60-9 2-9
EAC-Rosette (%) CSF Blood* 1 0 5 0 3 1
Null Cells (%) CSF Blood
5 10 5 19 5 1
39 53 25 31 57 30
7-6t
39-2
n.t.
1-7 0-82
39 52 36 12 29 30 n.t.
2-6
5.4
33-0 5.4
* Percentage of rosettes as determined by micromethod applied to corresponding samples of CSF and PBL. t Blood greater than CSF P< 0 1 (paired t-test analysis). n.t. = Not tested.
90
L. D. Wilkerson, R. P. Lisak & B. Zweiman
lymphocyte sub-populations in blood and CSF of individual animals showed poor correlation (r = 0. 16) even for the E-rosetting cells, despite their predominance in both compartments. The discrepancy between findings in CSF and blood was striking in terms of EAC-rosettes. They were infrequent or not identified in some samples of CSF despite reasonable prevalence in the corresponding blood sample. Because few EAC-rosetting lymphocytes were found in CSF, even when as many as 19% of peripheral blood mononuclear cells were of this type, additional studies were carried out to ascertain whether certain factors may have led to falsely low CSF levels. First, all available CSF lymphocytes (40,000 and 100,000) from two animals with EAE were used to determine EAC-rosette formation, and the results were still within the group variability. Secondly, aliquots of 7500 peripheral blood mononuclear cells from each of two normals were suspended in cell-free supernatant of CSF from two animals with EAE and incubated for 1 hr at 370C. The results of EAC-rosetting of mononuclear cells suspended in supernatant of CSF were compared to the results of micro and standard methods of EAC-rosette formation applied to other aliquots of the respective sample suspended in GVB. EAC-rosetting in the supernatant of CSF of animals with EAE gave results (6% and 1%) similar to those for matching aliquots rosetted in GVB (10% and 2%, respectively). DISCUSSION In 1951, Kabat et al. documented an increase in the proportion of gammaglobulin in the CSF protein of monkeys with EAE and noted, as had Rivers & Schwentker (1935), that samples of CSF from some, but not all, of the sick animals contained white cells. With this exception, we are not aware of prior notation of CSF pleocytosis during EAE, though occurrence of such a pleocytosis would be predictable from the findings of Waksman & Adams (1962) that a leptomeningeal round cell infiltrate was a prominent early pathological feature in most of their guinea-pigs. In the present study a moderate pleocytosis in CSF accompanied EAE in all neurologically sick animals. It was not found in CSF of any ofthe second group of animals studied at a time (9 or 10 days after sensitization) when previous work had shown that similarly injected animals manifested in vivo or in vitro evidence of sensitivity to the inducing antigen, although they still appeared healthy (Lisak & Zweiman, 1974a). The finding that virtually all cells in the pleocytosis were mononuclear parallels the absence of polymorphonuclear leucocytes and other cells from the round cell leptomeningeal infiltrate described by Waksman & Adams (1962). While the source of these cells found in the CSF of animals with EAE has yet to be defined, several potential means of accumulation can be discounted. First, these findings are unlikely to have been simply an artefact produced by the minimal contamination of the CSF by bleeding. Extrapolation from the ratios of RBC in CSF and the blood of individual animals suggests that only a very small number of leucocytes in CSF specimens could be explained by blood contamination. The difference between the proportion of lymphocytes found in CSF (mean 96.6%) and in blood (mean 27.6%) also discounts the likelihood that the pleocytosis observed should be attributed to an artefact of traumatic sampling. Further, the characteristics of the pleocytosis suggest that it is likewise not attributable to a diffuse or unselective alteration of the blood: CSF barrier. The lack of correlation between CSF and peripheral blood leucocyte counts, and the marked differences in differential counts in the respective compartments suggest a more selective process of cell accumulation. Although lymphocytes in both compartments were predominantly T-cells or null cells, there was a poor correlation between the proportions of T-cells and null cells in CSF and the blood of individual animals and a striking paucity of identified B-cells and monocytes in CSF. These differences are also more consistent with a process of selective cell accumulation. The constituency of the CSF pleocytosis in the animals with EAE is broadly consistent with that of a delayed hypersensitivity response although exactly what constitutes the latter varies with the animal species and with different models within the species, as emphasized by McCluskey & Leter (1974). Preliminary characterization of this pleocytosis suggests a parallel with the earlier observation of Waksman & Adams (1962) that most mononuclear cells in early lesions of EAE appeared to be lymphocytes. Whether the mononuclear cell subpopulations in CSF and within the parenchyma are parallel remains
CSF lymphocytes in EAE
91
to be shown. For the present, these observations suggest a compartmentalization of cells between CSF and blood during the course of a presumed delayed hypersensitivity response to a nervous system constituent, myelin basic protein. The question raised is whether and how such a compartmentalization might be related to the mounting ofthe hypersensitivity response. The superb help of Ms. Felicula Mercado is happily acknowledged. Dr P. Doherty taught us cisternal puncture methods which we adapted for this study. This study was supported by Grant No. 5 P20 NS11037-10 and the National Multiple Sclerosis Society Grant No. 894-B-2. REFERENCES
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