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Macrophage inflammatory protein-1α in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological diseases
JOURNAL
OF THE
NEUROLOGICAL SCIENCES
ELSEVIER
Journal of the Neurological Sciences 129 (1995) 223-227
Macrophage inflammatory protein-k in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological diseases Ryuji Miyagishi, Seiji Kikuchi Department of Neurology, Hokkaido
* , Toshiyuki Fukazawa, Kunio Tashiro
Universi@ School of Medicine, Kita 14 Nishi 5, Sapporo 060, Japan
Received 20 July 1994; revised 12 December 1994; accepted 19 December 1994
Abstract The level of macrophage inflammatory protein-lcu (MIP-la), a newly discovered cytokine of chemokine family, was determined in cerebrospinal fluid (CSF) from 18 patients with multiple sclerosis (MS) and from control patients with other neurological disorders by an enzyme-linked immunosorbent assay (ELISA). The concentration of MIP-la in CSF was significantly elevated in MS in relapse (4.4 pg/ml) compared with non-inflammatory neurological disease control samples (0.3 pg/ml) (p < 0.0002). These concentrations in MS patients correlated well with leukocyte cell counts and protein content in CSF (r = 0.845, p < 0.0001; r = 0.853, p < 0.0001, respectively). In other inflammatory neurological disorders such as BehGet’s disease and HTLV-1 associated myelopathy, significantly increased CSF levels of MIP-la were also observed. Chemokines are reported to play an important role in an early event of inflammation such as lymphocyte traffic. This report is the first study which confirmed the involvement of a chemokine in MS and other inflammatory neurological disorders. Keywords:
Macrophage
inflammatory
protein-la;
Chemokine;Multiple sclerosis;Cerebrospinalfluid
1. Introduction
Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system characterized by focal T cell and macrophage infiltration into white matter and demyelination. It is widely held that selective homing of lymphocytes to the central nervous system (CNS) represents an early feature of the inflammatory demyelinating lesion of experimental autoimmune encephalomyelitis (Raine et al., 1990). In a number of inflammatory responses, increasingly implicated are membrane interactions between homing receptors and adhesion molecules on endothelial cells. In addition, a newly recognized superfamily of chemoattractant cytokine, the chemokine, is also reported to play an important role in early events of inflammation (Wolpe et al., 1988; Wolpe and Cerami, 1989). Chemokines released by cells at sites of local tissue
* Corresponding author. Tel.: (+81-11) (+81-11)
716-1161 (6028); Fax:
757-2671.
0022-510X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022-510X(95)00004-6
damage have been recognized as important mediators of inflammation due to their ability to selectively enhance migration of specific leukocyte subsets. Chemokines compromise a superfamily of small, secreted proteins that mediate inflammation by inducing chemotaxis and activation of a variety of inflammatory cells. Based on the arrangement of the cystines, chemokine is divided into two subfamilies. C-X-C chemokines include interleukin(IL)-8, melanoma growth-stimulatory activity, platelet factor 4, p-thromboglobulin, and the C-C chemokines include macrophage inflammatory protein(MIP)-lcY, MIP-l@, monocyte chemotactic protein-l, RANTES (Wolpe and Cerami, 1989; Oppenheim et al., 1991). Chemokines, by definition, demonstrate chemotactic activity and a general pattern of chemokine-mediated chemotaxis has emerged as a rule: C-X-C chemokine attract neutrophils, but not macrophages, while C-C chemokines have no effects on neutrophils but do chemoattractant monocytes and T cells. Particular chemokines induce selective migration of leukocyte subset which differ both in phenotypic markers and activation state. For
224
R. Miyagishi et al. /Journal
of the Neurological
example, MIP-la at a low concentration attracts B cells and cytotoxic T cells, whereas at higher concentrations the migration of CD4 + T cells is enhanced. MIP-l@ selectively induces chemotaxis of activated CD4 + T lymphocytes (Schall et al., 1993; Taub et al., 1993). Differences in the kinetics of the expression between these chemokines may further coordinate the regulation of the migration pattern, and thus the composition of the lymphocyte population at inflammatory sites at any given time. In this study, we have examined MIP-la level in the CSF of MS patients, and investigated whether their presence correlates with clinical disease activity and immunological disturbance. This report is the first study, to our knowledge, which confirmed the involvement of a chemokine in MS and other inflammatory neurological disorders. 2. Materials
and methods
Patients We studied CSF samples from 18 patients (3 men and 15 women; 36.6 f 13.3 years, mean age + SD> with clinically definite MS according to the criteria of Poser et al. (1983). The median duration of illness was 7.8 years (range O-21 years). All had relapsing-remitting form of the disease; 13 were in acute relapse; and 5 had been in remission for at least 3 months. None had received steroids or other immunosuppressive drugs in the previous 3 months. CSF samples were obtained from 9 patients with other inflammatory neurological diseases (OIND). These included three neuro-Behqet’s disease, three HTLV-1 associated myelopathy (HAM), one aseptic meningitis and two CNS lymphoma. Eight patients with non-inflammatory neurological diseases (NIND) (43.6 + 9.2 years, mean age f SD> were included in the study to serve as neurological controls. Their diagnoses included two disk herniations, one motor neuron disease, one Parkinson’s disease, one spinocerebellar degeneration, one tension type headache, one myotonic dystrophy and one psychiatric disorder. CSF samples were obtained by non-traumatic lumbar puncture. We excluded xanthochromic CSF sam-
Sciences 129 (1995) 223-227
ples or samples containing more than 1 erythrocyte per ml at the time of collection. Total and differential cell counts were performed within 30 min using FuchsRosenthal chambers. IgG concentrations in unconcentrated CSF were determined by automated immunoprecipitation nephelometry. All samples were stored at - 80” C until use. MIP-1 (Y ELBA MIP-la measurements were carried out in blind fashion with an ELISA kit (Quantikine, R and D systems, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, it employs the quantitative “sandwich” enzyme immunoassay technique. A monoclonal antibody specific for MIP-la has been coated onto the microtiter plate provided in the kit. 200 ml of CSF samples 5-fold concentrated are pipetted into the wells in duplicate and any MIP-la presented is bound by the immobilized antibody. After washing away any unbound sample proteins, an enzyme linked polyclonal antibody specific for MIP-la is added to the wells to “sandwich” any MIP-la immobilized during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of MIP-la bound in the initial step. The color development is stopped and absorbance read at 450 nm with a blank reading at 540 nm. The detection limit for MIP-la was 0.4 pg/ml. We evaluated the correlation of MIP-la levels and mononuclear cell count or protein level in the CSF from MS patients. Statistics The non-parametric Mann-Whitney test was used for statistical analysis. The correlation coefficient r was defined by Pearson’s correlation matrix test. 3. Results
In the non-inflammatory neurological diseases (NIND) group, the mean + 3 SD (2.4 pg/ml) was calculated as the cut-off value for determining abnormally high levels of MIP-la in this study population.
Table 1 Clinical characteristics of patients and MIP-lc~ levels in CSF. The increase of MIP-la in MS was statistically significant (relapse non-inflammatory neurological diseases (NIND) controls, p < 0.0002; remission vs NIND controls, p < 0.007; Mann-Whitney U-test). No. of patients MS relapse MS remission NIND controls
**
13 5 8
* Significantly different from control group. * * NIND: non-inflammatory neurological disease.
Mean (SD) age (yrs)
Mean (SD) MIP-la
33.9 (13.2) 43.0 (12.2) 43.6 (9.2)
4.4 (5.0) 1.9 (0.9) 0.3 (0.7)
levels (pg/ml)
p values <0.0002 * 0.007 *
vs
R. Miyagishi et al. /Journal
of the Neurological Sciences 129 (1995) 223-227
225
w/ml 100 i
controls
MS
Other
Lymphoma
HAM
Behcet
Inflammatory
Meningitis
Neurological
Diseases
Fig. 1. MIP-la were detected in CSF from patients in MS relapse (62%) and in remission (20%), but not detected in non-inflammatory neurological diseases (NIND) control samples. The detection limit was 0.4 pg/ml. Several patients with BehGet’s disease, HTLV-1 associated myelopathy (HAM), and meningitis showed increased MIP-la levels in CSF. The horizontal dashed line is the upper limit of normal.
High MIP-la levels were detected in the CSF of 8 of 13 patients (62%) with an acute relapse of MS (4.4 + 5 pg/ml, mean k- SD), 1 of 5 patients (20%) in remission (1.9 k 0.9 pg/ml) and negative in NIND controls (0.3 f 0.7 pg/ml) (Table 1). CSF MIP-lcu concentrations of MS in both relapse and remission were
Fiq.2a
PST/ml 20
significantly higher than NIND controls (p = 0.0002, p = 0.007, respectively), whereas these concentrations failed to differentiate between MS patients in relapse and those in remission (p = 0.075). CSF samples from patients with other inflammatory neurological diseases(OIND) frequently exhibited high
Fiq.2b
pg/ml
1
20
40
60
80
/nun3
-
I
80
40
Mononuclear Fig. 2. a: significant correlation between MIP-la correlation matrix test). b: significant correlation Pearson’s correlation matrix test).
cells
120
w/d1
Protein
levels and mononuclear cell count in CSF from MS patients (r = 0.845, p < 0.0001, Pearson’s between MIP-lcr levels and protein content in CSF from MS patients (r = 0.853, p < 0.0001,
226
R. Miyagishi et al. /Journal
of the Neurological
MIP-la levels: 3 cases of 3 with neuro-Behqet’s disease, 2 of 3 with HAM, 1 of 2 CNS lymphoma and 1 of 1 aseptic meningitis (Fig. 1). CSF concentrations of MIP-la in MS patients correlated with CSF mononuclear cell count and CSF protein content (r = 0.845, p < 0.0001; r = 0.853, p < 0.0001, respectively) (Fig. 2), but they failed to correlate with intrathecal IgG levels. One MS patient with high levels of MIP-lc~ (19.5 pg/ml) had transverse myelopathy and optic neuritis during the course of illness; but, in general, MIP-la levels in CSF did not correlate with the clinical forms (for example, cerebral vs spinal form) or total disease duration. This study demonstrated an elevation of MIP-la concentrations in the CSF in MS and other inflammatory neurological diseases (OIND) patients. Chemokines have been increasingly recognized as important immune mediators involved in the pathogenesis of variety of inflammatory disease states (Wolpe and Cerami, 1989). A model of adhesion cascade has been proposed to explain the adhesion of many leukocytes subtypes to endothelium (Rot, 1992; Tanaka et al., 1993). Circulating cells are first loosely ‘tethered’ to endothelium by selectin-mediated adhesion. This would allow presentation of proteoglycan-bound chemokine by endothelial cells to its receptor on T cells. The bound chemokine triggers functional activation of the leukocyte integrins, and cells can migrate into tissue under the influence of local chemotactic factors. In several studies of lung inflammatory disorders, the content of MIP-la in bronchial fluid was increased and its disposition was demonstrated with an immunohistochemical technique (Standiford et al., 1993). We also have some evidence of MIP-la deposition on the surface of endothelial cells and extra cellular matrix of MS brain from immunohistochemical studies (unpublished). There is the possibility that MIP-la has several isoforms and that some of them are not bound but soluble in CSF and serum, since soluble forms have been demonstrated on several cytokine receptors and adhesion molecules. We are now investigating this possibility using immunoblotting of concentrated CSF. The concentrations of MIP-la in the CSF of inflammatory neurological diseases were less than 50 pg/ml, which raises the question of this being too low to be significant. Many studies have been performed to detect the cytokines, for example tumor necrosis factor (TNF), IL-l, IL-2, IL-6 in the CSF of MS patients but the results are widely variable (Maimone et al., 1991; Peter et al., 1991; Sharief and Hentges, 1991; Tsukada et al., 1991). As for TNF, Tsukada et al. (1991) found elevated levels of TNF, the highest value was about 25 pg/ml, while Maimone et al. (1991) reported TNF levels between 40 pg/ml and 150 pg/ml. Although we can not directly compare their values with our data because of the differences in molecular weights and
Sciences I29 (1995) 223-227
biological potencies, we propose that the involvement of MIP-la may be significant in the diseases mentioned above. In all the inflammatory neurological diseases studied, CSF MIP-la was similarly elevated, limiting its usefulness as a diagnostic tool. We concentrated our analyses on MS, in which differences between stages were evident. IL-2 and TNF were reported to be elevated in the CSF of MS patients, especially in the acute relapse stage (Tsukada et al., 1991; Sharief and Thompson, 1992,1993). Similarly, CSF MIP-la was elevated in the acute relapse stage. Although the IL-2 concentration in CSF was shown to correlate with the duration of illness or disease activity (Sharief and Thompson, 1993), there was no correlation between the concentration of MIP-la and duration of the illness. Nor could we demonstrate a correlation between CSF MIP-lcu concentrations and the clinical forms. In one case, a slight amount of MIP-la was detected in CSF even during remission. We proposed that this patient seemed to enter into a secondary progressive clinical course. We suspect that there was some predisposition for lymphocytes, and that once activated, they can immediately and easily trespass into the brain parenchyma along the guidance of MIP-la. We analyzed the relationship of MIP-la content with other CSF markers. First, the leukocyte count in CSF was shown to be strongly correlated with MIP-lcr concentration in MS patients. Are these cells producing MIP-la in CSF? Can these cells respond to MIP-lcu with its receptors on them? Furthermore, each member of the chemokine family has a subtype pattern of cells responsive to it. The CSF protein concentration was also correlated with MIP-la concentration in MS patients. Since the protein content in CSF is thought to be a marker of permeability of brain vessels, the increased MIP-la may derive from serum. Otherwise, it may be synthesized within CNS. To answer this, one must analyze the index similar to IgG index. Since we did not elucidate any correlation between MIP-la content and IgG in CSF, the elevation of MIP-la did not influence the activation of B cells in MS. In another disease such as neuro-Behqet’s disease, MIP-la was markedly elevated in CSF. Recently it was reported that MIP-lcu is also synthesized by polymorphonuclear cells (Kasama et al., 19931,whose CSF count is increased in Behset’s disease. In HAM, the factors involved in T cell traffic and its persistence within CNS are intriguing. Although T cells infiltrating into the CNS of HAM patients do not reactive to CNS antigens in contrast with T cells in MS patients, which are reactive to CNS antigens such as myelin basic protein and/or proteolipid protein, they are retained within the CNS. MIPla may play some role in this. This work was supported by a Grant-in-Aid for
R. Miyagishi et al. /Journal
of the Neurological
Scientific Research from the Ministry of Education, Science and Culture of Japan, 05454257.
References Kasama, T., R.M. Strieter, T.J. Standiford, M.D. Burdick and S.L. Kunkel (1993) Expression and regulation of human neutrophilderived macrophage inflammatory protein-la. J. Exp. Med., 178: 63-72. Maimone, D., S. Gregory, B.G.W. Arnason and A.T. Reder (1991) Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis. J. Neuroimmunol., 32: 67-74. Oppenheim, J.J., C.O.C. Zachariae, N. Mukaida and K. Matsushima (1991) Properties of the novel proinflammatory supergene “intercrine” cytokine family. Annu. Rev. Immunol., 9: 617-648. Peter, J.B., F.N. Boctor and W.W. Tourtellotte (1991) Serum and CSF levels of IL-2, sIL-2R, TNFa and IL-lp in chronic progressive multiple sclerosis: expected lack of clinical utility. Neurology, 37: 1097-l 102. Poser, CM., D.W. Paty, L. Scheinberg, WI. McDonald, F.A. Davis, G. Ebers, K.P. Johnson, W.A. Sibley, D.H. Silberberg and W.W. Tourtellotte (1983) New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol., 13: 227-231. Raine, C.S., B. Bannella, A.M. Duijvestijn and A.H. Cross (1990) Homing to central nervous system vasculature by antigen-specific lymphocytes. II. Lymphocyte/endothelial cell adhesion during the initial stages of autoimmune demyelination. Lab. Invest., 63: 476-489. Rot, A. (1992) Endothelial cell binding of NAP-l/IL-8: role in neutrophil emigration. Immunol. Today, 13: 291-294. Schall, T.J., K. Bacon, R.D.R. Camp, J.W. Kaspari and D.V. Goed-
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227
del (1993) Human macrophage inflammatory protein a (MIP-la) and MIP-1P chemokines attract distinct populations of lymphocytes. J. Exp. Med., 177: 1821-1826. Sharief, M.K. and R. Hentges (1991) Association between tumor necrosis factora and disease progression in patients with multiple sclerosis. N. Engl. J. Med., 325: 467-472. Sharief, M.K. and E.J. Thompson (1992) In vivo relationship of tumor necrosis factor* to blood-brain barrier damage in patients with active multiple sclerosis. J. Neuroimmunol., 38: 27-34. Sharief, M.K. and E.J. Thompson (1993) Correlation of interleukin-2 and soluble interleukin-2 receptor with clinical activity of multiple sclerosis. J. Neurol. Neurosurg. Psychiat., 56: 169-174. Standiford, T.J., M.W. Rolfe, S.L. Kunkel, J.P. Lynch, M.D. Burdick, A.R. Gilbert, M.B. Orringer, R.I. Whyte and R.M. Strieter (19931 Macrophage inflammatory protein-la expression in interstitial lung disease. J. Immunol., 151: 2852-2863. Tanaka, Y., D.H. Adams, S. Hubscher, H. Hirano, U. Siebenlist and S. Shaw (1993) T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1P. Nature, 361: 79-82. Taub, D.D., K. Conlon, A.R. Lloyd, J.J. Oppenheim and D.J. Kelvin (1993) Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-la and MIP-l/3. Science, 260: 355-358. Tsukada, N., K. Miyagi, M. Matsuda, N. Yanagisawa and K. Yone (1991) Tumor necrosis factor and interleukin-1 in the CSF and sera of patients with multiple sclerosis. J. Neurol. Sci., 104: 230-234. Wolpe, S.D., G. Davatelis, B.B. Sherry, B. Beutler, D.G. Hesse, H.T. Nguyen, L.L. Moldawer, C.F. Nathan, S.F. Lowry and A. Cerami (1988) Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J. Exp. Med., 167: 570-581. Wolpe, S.D. and A. Cerami (1989) Macrophage inflammatory protein 1 and 2: members of a novel superfamily of cytokines. FASEB J., 3: 2565-2573.
OF THE
NEUROLOGICAL SCIENCES
ELSEVIER
Journal of the Neurological Sciences 129 (1995) 223-227
Macrophage inflammatory protein-k in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological diseases Ryuji Miyagishi, Seiji Kikuchi Department of Neurology, Hokkaido
* , Toshiyuki Fukazawa, Kunio Tashiro
Universi@ School of Medicine, Kita 14 Nishi 5, Sapporo 060, Japan
Received 20 July 1994; revised 12 December 1994; accepted 19 December 1994
Abstract The level of macrophage inflammatory protein-lcu (MIP-la), a newly discovered cytokine of chemokine family, was determined in cerebrospinal fluid (CSF) from 18 patients with multiple sclerosis (MS) and from control patients with other neurological disorders by an enzyme-linked immunosorbent assay (ELISA). The concentration of MIP-la in CSF was significantly elevated in MS in relapse (4.4 pg/ml) compared with non-inflammatory neurological disease control samples (0.3 pg/ml) (p < 0.0002). These concentrations in MS patients correlated well with leukocyte cell counts and protein content in CSF (r = 0.845, p < 0.0001; r = 0.853, p < 0.0001, respectively). In other inflammatory neurological disorders such as BehGet’s disease and HTLV-1 associated myelopathy, significantly increased CSF levels of MIP-la were also observed. Chemokines are reported to play an important role in an early event of inflammation such as lymphocyte traffic. This report is the first study which confirmed the involvement of a chemokine in MS and other inflammatory neurological disorders. Keywords:
Macrophage
inflammatory
protein-la;
Chemokine;Multiple sclerosis;Cerebrospinalfluid
1. Introduction
Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system characterized by focal T cell and macrophage infiltration into white matter and demyelination. It is widely held that selective homing of lymphocytes to the central nervous system (CNS) represents an early feature of the inflammatory demyelinating lesion of experimental autoimmune encephalomyelitis (Raine et al., 1990). In a number of inflammatory responses, increasingly implicated are membrane interactions between homing receptors and adhesion molecules on endothelial cells. In addition, a newly recognized superfamily of chemoattractant cytokine, the chemokine, is also reported to play an important role in early events of inflammation (Wolpe et al., 1988; Wolpe and Cerami, 1989). Chemokines released by cells at sites of local tissue
* Corresponding author. Tel.: (+81-11) (+81-11)
716-1161 (6028); Fax:
757-2671.
0022-510X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022-510X(95)00004-6
damage have been recognized as important mediators of inflammation due to their ability to selectively enhance migration of specific leukocyte subsets. Chemokines compromise a superfamily of small, secreted proteins that mediate inflammation by inducing chemotaxis and activation of a variety of inflammatory cells. Based on the arrangement of the cystines, chemokine is divided into two subfamilies. C-X-C chemokines include interleukin(IL)-8, melanoma growth-stimulatory activity, platelet factor 4, p-thromboglobulin, and the C-C chemokines include macrophage inflammatory protein(MIP)-lcY, MIP-l@, monocyte chemotactic protein-l, RANTES (Wolpe and Cerami, 1989; Oppenheim et al., 1991). Chemokines, by definition, demonstrate chemotactic activity and a general pattern of chemokine-mediated chemotaxis has emerged as a rule: C-X-C chemokine attract neutrophils, but not macrophages, while C-C chemokines have no effects on neutrophils but do chemoattractant monocytes and T cells. Particular chemokines induce selective migration of leukocyte subset which differ both in phenotypic markers and activation state. For
224
R. Miyagishi et al. /Journal
of the Neurological
example, MIP-la at a low concentration attracts B cells and cytotoxic T cells, whereas at higher concentrations the migration of CD4 + T cells is enhanced. MIP-l@ selectively induces chemotaxis of activated CD4 + T lymphocytes (Schall et al., 1993; Taub et al., 1993). Differences in the kinetics of the expression between these chemokines may further coordinate the regulation of the migration pattern, and thus the composition of the lymphocyte population at inflammatory sites at any given time. In this study, we have examined MIP-la level in the CSF of MS patients, and investigated whether their presence correlates with clinical disease activity and immunological disturbance. This report is the first study, to our knowledge, which confirmed the involvement of a chemokine in MS and other inflammatory neurological disorders. 2. Materials
and methods
Patients We studied CSF samples from 18 patients (3 men and 15 women; 36.6 f 13.3 years, mean age + SD> with clinically definite MS according to the criteria of Poser et al. (1983). The median duration of illness was 7.8 years (range O-21 years). All had relapsing-remitting form of the disease; 13 were in acute relapse; and 5 had been in remission for at least 3 months. None had received steroids or other immunosuppressive drugs in the previous 3 months. CSF samples were obtained from 9 patients with other inflammatory neurological diseases (OIND). These included three neuro-Behqet’s disease, three HTLV-1 associated myelopathy (HAM), one aseptic meningitis and two CNS lymphoma. Eight patients with non-inflammatory neurological diseases (NIND) (43.6 + 9.2 years, mean age f SD> were included in the study to serve as neurological controls. Their diagnoses included two disk herniations, one motor neuron disease, one Parkinson’s disease, one spinocerebellar degeneration, one tension type headache, one myotonic dystrophy and one psychiatric disorder. CSF samples were obtained by non-traumatic lumbar puncture. We excluded xanthochromic CSF sam-
Sciences 129 (1995) 223-227
ples or samples containing more than 1 erythrocyte per ml at the time of collection. Total and differential cell counts were performed within 30 min using FuchsRosenthal chambers. IgG concentrations in unconcentrated CSF were determined by automated immunoprecipitation nephelometry. All samples were stored at - 80” C until use. MIP-1 (Y ELBA MIP-la measurements were carried out in blind fashion with an ELISA kit (Quantikine, R and D systems, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, it employs the quantitative “sandwich” enzyme immunoassay technique. A monoclonal antibody specific for MIP-la has been coated onto the microtiter plate provided in the kit. 200 ml of CSF samples 5-fold concentrated are pipetted into the wells in duplicate and any MIP-la presented is bound by the immobilized antibody. After washing away any unbound sample proteins, an enzyme linked polyclonal antibody specific for MIP-la is added to the wells to “sandwich” any MIP-la immobilized during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of MIP-la bound in the initial step. The color development is stopped and absorbance read at 450 nm with a blank reading at 540 nm. The detection limit for MIP-la was 0.4 pg/ml. We evaluated the correlation of MIP-la levels and mononuclear cell count or protein level in the CSF from MS patients. Statistics The non-parametric Mann-Whitney test was used for statistical analysis. The correlation coefficient r was defined by Pearson’s correlation matrix test. 3. Results
In the non-inflammatory neurological diseases (NIND) group, the mean + 3 SD (2.4 pg/ml) was calculated as the cut-off value for determining abnormally high levels of MIP-la in this study population.
Table 1 Clinical characteristics of patients and MIP-lc~ levels in CSF. The increase of MIP-la in MS was statistically significant (relapse non-inflammatory neurological diseases (NIND) controls, p < 0.0002; remission vs NIND controls, p < 0.007; Mann-Whitney U-test). No. of patients MS relapse MS remission NIND controls
**
13 5 8
* Significantly different from control group. * * NIND: non-inflammatory neurological disease.
Mean (SD) age (yrs)
Mean (SD) MIP-la
33.9 (13.2) 43.0 (12.2) 43.6 (9.2)
4.4 (5.0) 1.9 (0.9) 0.3 (0.7)
levels (pg/ml)
p values <0.0002 * 0.007 *
vs
R. Miyagishi et al. /Journal
of the Neurological Sciences 129 (1995) 223-227
225
w/ml 100 i
controls
MS
Other
Lymphoma
HAM
Behcet
Inflammatory
Meningitis
Neurological
Diseases
Fig. 1. MIP-la were detected in CSF from patients in MS relapse (62%) and in remission (20%), but not detected in non-inflammatory neurological diseases (NIND) control samples. The detection limit was 0.4 pg/ml. Several patients with BehGet’s disease, HTLV-1 associated myelopathy (HAM), and meningitis showed increased MIP-la levels in CSF. The horizontal dashed line is the upper limit of normal.
High MIP-la levels were detected in the CSF of 8 of 13 patients (62%) with an acute relapse of MS (4.4 + 5 pg/ml, mean k- SD), 1 of 5 patients (20%) in remission (1.9 k 0.9 pg/ml) and negative in NIND controls (0.3 f 0.7 pg/ml) (Table 1). CSF MIP-lcu concentrations of MS in both relapse and remission were
Fiq.2a
PST/ml 20
significantly higher than NIND controls (p = 0.0002, p = 0.007, respectively), whereas these concentrations failed to differentiate between MS patients in relapse and those in remission (p = 0.075). CSF samples from patients with other inflammatory neurological diseases(OIND) frequently exhibited high
Fiq.2b
pg/ml
1
20
40
60
80
/nun3
-
I
80
40
Mononuclear Fig. 2. a: significant correlation between MIP-la correlation matrix test). b: significant correlation Pearson’s correlation matrix test).
cells
120
w/d1
Protein
levels and mononuclear cell count in CSF from MS patients (r = 0.845, p < 0.0001, Pearson’s between MIP-lcr levels and protein content in CSF from MS patients (r = 0.853, p < 0.0001,
226
R. Miyagishi et al. /Journal
of the Neurological
MIP-la levels: 3 cases of 3 with neuro-Behqet’s disease, 2 of 3 with HAM, 1 of 2 CNS lymphoma and 1 of 1 aseptic meningitis (Fig. 1). CSF concentrations of MIP-la in MS patients correlated with CSF mononuclear cell count and CSF protein content (r = 0.845, p < 0.0001; r = 0.853, p < 0.0001, respectively) (Fig. 2), but they failed to correlate with intrathecal IgG levels. One MS patient with high levels of MIP-lc~ (19.5 pg/ml) had transverse myelopathy and optic neuritis during the course of illness; but, in general, MIP-la levels in CSF did not correlate with the clinical forms (for example, cerebral vs spinal form) or total disease duration. This study demonstrated an elevation of MIP-la concentrations in the CSF in MS and other inflammatory neurological diseases (OIND) patients. Chemokines have been increasingly recognized as important immune mediators involved in the pathogenesis of variety of inflammatory disease states (Wolpe and Cerami, 1989). A model of adhesion cascade has been proposed to explain the adhesion of many leukocytes subtypes to endothelium (Rot, 1992; Tanaka et al., 1993). Circulating cells are first loosely ‘tethered’ to endothelium by selectin-mediated adhesion. This would allow presentation of proteoglycan-bound chemokine by endothelial cells to its receptor on T cells. The bound chemokine triggers functional activation of the leukocyte integrins, and cells can migrate into tissue under the influence of local chemotactic factors. In several studies of lung inflammatory disorders, the content of MIP-la in bronchial fluid was increased and its disposition was demonstrated with an immunohistochemical technique (Standiford et al., 1993). We also have some evidence of MIP-la deposition on the surface of endothelial cells and extra cellular matrix of MS brain from immunohistochemical studies (unpublished). There is the possibility that MIP-la has several isoforms and that some of them are not bound but soluble in CSF and serum, since soluble forms have been demonstrated on several cytokine receptors and adhesion molecules. We are now investigating this possibility using immunoblotting of concentrated CSF. The concentrations of MIP-la in the CSF of inflammatory neurological diseases were less than 50 pg/ml, which raises the question of this being too low to be significant. Many studies have been performed to detect the cytokines, for example tumor necrosis factor (TNF), IL-l, IL-2, IL-6 in the CSF of MS patients but the results are widely variable (Maimone et al., 1991; Peter et al., 1991; Sharief and Hentges, 1991; Tsukada et al., 1991). As for TNF, Tsukada et al. (1991) found elevated levels of TNF, the highest value was about 25 pg/ml, while Maimone et al. (1991) reported TNF levels between 40 pg/ml and 150 pg/ml. Although we can not directly compare their values with our data because of the differences in molecular weights and
Sciences I29 (1995) 223-227
biological potencies, we propose that the involvement of MIP-la may be significant in the diseases mentioned above. In all the inflammatory neurological diseases studied, CSF MIP-la was similarly elevated, limiting its usefulness as a diagnostic tool. We concentrated our analyses on MS, in which differences between stages were evident. IL-2 and TNF were reported to be elevated in the CSF of MS patients, especially in the acute relapse stage (Tsukada et al., 1991; Sharief and Thompson, 1992,1993). Similarly, CSF MIP-la was elevated in the acute relapse stage. Although the IL-2 concentration in CSF was shown to correlate with the duration of illness or disease activity (Sharief and Thompson, 1993), there was no correlation between the concentration of MIP-la and duration of the illness. Nor could we demonstrate a correlation between CSF MIP-lcu concentrations and the clinical forms. In one case, a slight amount of MIP-la was detected in CSF even during remission. We proposed that this patient seemed to enter into a secondary progressive clinical course. We suspect that there was some predisposition for lymphocytes, and that once activated, they can immediately and easily trespass into the brain parenchyma along the guidance of MIP-la. We analyzed the relationship of MIP-la content with other CSF markers. First, the leukocyte count in CSF was shown to be strongly correlated with MIP-lcr concentration in MS patients. Are these cells producing MIP-la in CSF? Can these cells respond to MIP-lcu with its receptors on them? Furthermore, each member of the chemokine family has a subtype pattern of cells responsive to it. The CSF protein concentration was also correlated with MIP-la concentration in MS patients. Since the protein content in CSF is thought to be a marker of permeability of brain vessels, the increased MIP-la may derive from serum. Otherwise, it may be synthesized within CNS. To answer this, one must analyze the index similar to IgG index. Since we did not elucidate any correlation between MIP-la content and IgG in CSF, the elevation of MIP-la did not influence the activation of B cells in MS. In another disease such as neuro-Behqet’s disease, MIP-la was markedly elevated in CSF. Recently it was reported that MIP-lcu is also synthesized by polymorphonuclear cells (Kasama et al., 19931,whose CSF count is increased in Behset’s disease. In HAM, the factors involved in T cell traffic and its persistence within CNS are intriguing. Although T cells infiltrating into the CNS of HAM patients do not reactive to CNS antigens in contrast with T cells in MS patients, which are reactive to CNS antigens such as myelin basic protein and/or proteolipid protein, they are retained within the CNS. MIPla may play some role in this. This work was supported by a Grant-in-Aid for
R. Miyagishi et al. /Journal
of the Neurological
Scientific Research from the Ministry of Education, Science and Culture of Japan, 05454257.
References Kasama, T., R.M. Strieter, T.J. Standiford, M.D. Burdick and S.L. Kunkel (1993) Expression and regulation of human neutrophilderived macrophage inflammatory protein-la. J. Exp. Med., 178: 63-72. Maimone, D., S. Gregory, B.G.W. Arnason and A.T. Reder (1991) Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis. J. Neuroimmunol., 32: 67-74. Oppenheim, J.J., C.O.C. Zachariae, N. Mukaida and K. Matsushima (1991) Properties of the novel proinflammatory supergene “intercrine” cytokine family. Annu. Rev. Immunol., 9: 617-648. Peter, J.B., F.N. Boctor and W.W. Tourtellotte (1991) Serum and CSF levels of IL-2, sIL-2R, TNFa and IL-lp in chronic progressive multiple sclerosis: expected lack of clinical utility. Neurology, 37: 1097-l 102. Poser, CM., D.W. Paty, L. Scheinberg, WI. McDonald, F.A. Davis, G. Ebers, K.P. Johnson, W.A. Sibley, D.H. Silberberg and W.W. Tourtellotte (1983) New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol., 13: 227-231. Raine, C.S., B. Bannella, A.M. Duijvestijn and A.H. Cross (1990) Homing to central nervous system vasculature by antigen-specific lymphocytes. II. Lymphocyte/endothelial cell adhesion during the initial stages of autoimmune demyelination. Lab. Invest., 63: 476-489. Rot, A. (1992) Endothelial cell binding of NAP-l/IL-8: role in neutrophil emigration. Immunol. Today, 13: 291-294. Schall, T.J., K. Bacon, R.D.R. Camp, J.W. Kaspari and D.V. Goed-
Sciences 129 (I 995) 223-227
227
del (1993) Human macrophage inflammatory protein a (MIP-la) and MIP-1P chemokines attract distinct populations of lymphocytes. J. Exp. Med., 177: 1821-1826. Sharief, M.K. and R. Hentges (1991) Association between tumor necrosis factora and disease progression in patients with multiple sclerosis. N. Engl. J. Med., 325: 467-472. Sharief, M.K. and E.J. Thompson (1992) In vivo relationship of tumor necrosis factor* to blood-brain barrier damage in patients with active multiple sclerosis. J. Neuroimmunol., 38: 27-34. Sharief, M.K. and E.J. Thompson (1993) Correlation of interleukin-2 and soluble interleukin-2 receptor with clinical activity of multiple sclerosis. J. Neurol. Neurosurg. Psychiat., 56: 169-174. Standiford, T.J., M.W. Rolfe, S.L. Kunkel, J.P. Lynch, M.D. Burdick, A.R. Gilbert, M.B. Orringer, R.I. Whyte and R.M. Strieter (19931 Macrophage inflammatory protein-la expression in interstitial lung disease. J. Immunol., 151: 2852-2863. Tanaka, Y., D.H. Adams, S. Hubscher, H. Hirano, U. Siebenlist and S. Shaw (1993) T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1P. Nature, 361: 79-82. Taub, D.D., K. Conlon, A.R. Lloyd, J.J. Oppenheim and D.J. Kelvin (1993) Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-la and MIP-l/3. Science, 260: 355-358. Tsukada, N., K. Miyagi, M. Matsuda, N. Yanagisawa and K. Yone (1991) Tumor necrosis factor and interleukin-1 in the CSF and sera of patients with multiple sclerosis. J. Neurol. Sci., 104: 230-234. Wolpe, S.D., G. Davatelis, B.B. Sherry, B. Beutler, D.G. Hesse, H.T. Nguyen, L.L. Moldawer, C.F. Nathan, S.F. Lowry and A. Cerami (1988) Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J. Exp. Med., 167: 570-581. Wolpe, S.D. and A. Cerami (1989) Macrophage inflammatory protein 1 and 2: members of a novel superfamily of cytokines. FASEB J., 3: 2565-2573.