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M S Freedman

Publications and source records attributed to M S Freedman.

At least 19 recordsLinked to original sources
PubMed1998

Mechanism of gammadelta T cell-induced human oligodendrocyte cytotoxicity: relevance to multiple sclerosis.

Gammadelta T cells may contribute to the pathogenesis of Multiple Sclerosis (MS) via cytotoxicity directed at the myelin-oligodendrocyte unit. We have previously demonstrated that peripheral blood-derived gammadelta T cells lyse fresh human oligodendrocytes in vitro. The present work extends these observations to gammadelta T cells derived from both peripheral blood (PBL) and cerebrospinal fluid (CSF) of MS and non-MS neurological disease controls and addresses the mechanism of cellular cytotoxicity. We found that MS patients contained increased proportions of Vdelta1+ gammadelta T cells in both CSF and PBL samples compared to other neurological disease (OND) controls. Although gammadelta T cells from all patients were cytotoxic towards Daudi, RPMI 8226, U937, Jurkat, oligodendroglioma and fresh human oligodendrocyte targets, OND-derived, Vdelta2+ rich, populations derived from the CSF exhibited greater cytotoxicity towards cell lines (Daudi, RPMI 8226) known to express high levels of heat shock proteins (hsp). To clarify the mechanism(s) of cytotoxicity used by gammadelta T cells, we first showed that cell-target contact was necessary by the use of physical barriers (transwells), which reduced target cell lysis by at least 75%. The use of Ca2+-free media reduced lysis by up to 50%, but fully blocking gammadelta T cell Perforin release and function by either Ca2+ chelation (Mg2EGTA) or the H+-ATPase inhibitor Concanamycin-A (CMA), completely abrogated the lysis of Fas-/hsp60high expressing targets (Daudi, U937). However, additional treatment with Brefeldin A was required for the complete inhibition of gammadelta T cell mediated killing of Fas+ expressing Jurkat targets and fresh human brain-derived oligodendrocytes. Inhibition of granzyme activity by an isocoumarin compound reduced cytolysis only slightly. The use of either Brefeldin A or an anti-Fas antibody alone did not significantly affect lysis. These findings suggest that in MS, gammadelta T cells may utilize either the Fas-mediated or Perforin-based cell cytotoxicity pathways in exerting oligodendrocyte damage, though the Perforin pathway is predominant.

Brain↗
PubMed1997

Prostaglandin D synthase concentration in cerebrospinal fluid and serum of patients with neurological disorders.

Prostaglandin D synthase (PGD synthase) or beta-trace protein is a major constituent of human cerebrospinal fluid (CSF) representing-3% of the total CSF protein. We have recently developed a highly specific immunofluorometric assay for PGD synthase, which enabled us to quantify the presence of PGD synthase in fluids and tissues not associated with the CNS. In this report we provide quantitative data of the presence of PGD synthase in CSF and serum from 302 subjects with various neurological diseases and symptoms. PGD synthase levels in CSF are approximately 35-fold higher than those of serum, with a median concentration of 11,299 micrograms/L. A statistically significant association of PGD synthase concentration in CSF was observed with both patient age and gender. There was no correlation between PGD synthase concentration in serum and patient age or gender. To evaluate the clinical utility of PGD synthase in diagnosing neurological diseases, the distribution pattern of PGD synthase in CSF and serum was examined for each neuropathology of 268 patients whose diagnosis was known. No statistical difference was observed between PGD synthase concentration in the CSF (129 cases) or the serum (94 cases) of multiple sclerosis afflicted subjects in comparison to all other patients studied. The distribution pattern was also not different for PGD synthase levels in CSF of patients with HIV/AIDS related neuropathies, viral meningitis and fibromyalgia. We conclude that PGD synthase measurement presents no clinical utility in diagnosing neurological disorders in adulthood. PGD synthase may have a physiological and/or pathological role in the developing brain and in neurodegenerative diseases.

Adolescent↗
PubMed1997

gamma delta T-cell-human glial cell interactions. I. In vitro induction of gammadelta T-cell expansion by human glial cells.

gamma delta T-cells are found in increased proportion in multiple sclerosis (MS) white matter plaque infiltrates compared with peripheral blood or spleen, raising the possibility that they are either specifically attracted to lesion sites or, once present, are stimulated to expand. We have previously shown that human oligodendrocytes (OGC) preferentially express heat shock proteins (hsp), molecules to which gamma delta T-cells have been known to react and that in vitro expanded gamma delta T-cells can lyse OGC. We therefore investigated whether human glial cells, that differentially express hsp, could stimulate gamma delta T-cell expansion from peripheral blood. We compared the glial cell-induced expansion to cell lines which also differentially express hsp and have been shown to selectively stimulate gamma delta T-cell expansion (e.g. RPMI 8226, Daudi). We found that both OGC and human fetal astrocytes (hFA) expressed hsp and stimulated the preferential expansion of gamma delta T-cells to about the same extent as the hsp expressing cell lines RPMI 8226 or Daudi, in the presence of exogenous interleukin-2 (IL-2) but without any T-cell mitogen. Furthermore, the type of gamma delta T-cells expanded were of the V delta 2 subtype known to be particularly reactive to hsp. Microglia, U937 cell lines or purified myelin membranes, which express little or no hsp, did not support gamma delta T-cell growth. These results therefore suggest that OGC may contribute to the local expansion of gamma delta T-cells within MS plaques. Potential harmful effects of gamma delta T-cells on OGC may thereby contribute to the immunopathogenesis of MS demyelination.

Astrocytes↗
PubMed1997

gamma delta T-cell-human glial cell interactions. II. Relationship between heat shock protein expression and susceptibility to cytolysis.

gamma delta T-cells have been implicated in the immunopathogenesis of multiple sclerosis (MS), possibly through interaction with heat shock proteins (hsp). We have previously demonstrated that human oligodendrocytes (OGC) express hsp on their surface and induce the proliferation and expansion of gamma delta T-cells. We also showed that gamma delta T-cells are highly cytolytic to OGC in vitro. The current study addresses whether gamma delta T-cell-induced cytotoxicity to OGC involves the recognition of hsp on OGC or some other ligand. We first compared the lytic potential for different human glial cells and found that gamma delta T-cells lysed OGC, microglia and human fetal astrocytes to the same extent, despite the preferential expression of hsp only on OGC. This suggested that either hsp was not involved in cytolytic recognition or that more than one ligand exists. To address this we used cell lines that either shared OGC properties of hsp expression and the ability to stimulate gamma delta T-cells (RPMI 8226, Daudi) or did not (U937) in cold target competition assays with OGC. Results demonstrated that although all the cell lines were effectively killed by gamma delta T-cells, only the RPMI 8226 and Daudi cells were able to effectively compete for lysis with the OGC. These results support the notion that probably more than one ligand for gamma delta T-cell cytotoxic recognition exists but hsp could still be involved in gamma delta T-cell-induced lysis of OGC. Regulating the expression of hsp on OGC might therefore be a way of interfering with potential gamma delta T-cell-induced damage in MS.

Astrocytes↗
PubMed1997

Failure of intravenous immunoglobulin to arrest progression of multiple sclerosis: a clinical and MRI based study.

Due to the modest benefit, inconvenience and high cost of currently available therapies for MS, it is appropriate to seek alternative treatments. Based on anecdotal evidence suggestive of benefit for i.v.IG in MS, we conducted an open-label, unblinded protocol of i.v.IG in nine MS patients. The patients were given induction doses of i.v.IG followed by monthly boosters for 1 year and had clinical, MRI and CSF analyses performed. Patients included were both progressive and relapsing. There was no clinical benefit nor apparent MRI benefit utilizing this protocol. During treatment the majority of patients continued to progress or have attacks and MRI demonstrated continued accumulation of T2-weighted lesions. CSF was unaffected by treatment.

Adult↗
PubMed1994

Cytokine induction of heat shock protein expression in human oligodendrocytes: an interleukin-1-mediated mechanism.

In this study, we examined the role of cytokines, known to be in elevated levels in multiple sclerosis (MS) plaques, in regulating oligodendrocyte (ODC) expression of heat shock protein (hsp) in human brain-derived glial cell cultures. Using dual-stain immunohistochemistry, we initially compared the ability of a mixture of cytokines (IL-1 alpha, IL-1 beta, IL-2, IL-6, IL-8, TNF-alpha, TNF-beta, IFN-beta and IFN-gamma) with that of physical stimuli such as heat shock and peroxide, to increase cellular expression of the mainly inducible hsp72 species in mixed glial cell cultures (containing ODC, astrocytes and microglia). Similar to heat shock and peroxide, the cytokine mixture induced hsp72 expression only in ODC (70 +/- 5% vs. a baseline of 3 +/- 1% positive cells). When used individually, however, only IL-1 alpha (79 +/- 3%), IFN-gamma (70 +/- 2%) and TNF-alpha (65 +/- 5%) induced ODC hsp72 expression in mixed glial cell cultures. In purified ODC preparations, only IL-1 alpha induced hsp72 expression (84 +/- 4%). An IL-1 receptor antagonist (IL-1ra), abrogated hsp72 induction by IL-1 alpha (16 +/- 3%) as well as that due to IFN-gamma (14 +/- 1%) and TNF-alpha (13 +/- 2%) in mixed glial cell cultures. Furthermore, ODC express IL-1 receptors, detected by confocal laser scanning microscopy. Our data indicate that cytokines mediate hsp induction in ODC possibly via a final common pathway involving IL-1 binding to its receptor on ODC. Such interaction could enhance any putative ODC-immune interactions which are dependent on hsp molecule recognition.

Animals↗
PubMed1994

Heat shock protein immunoreactivity in CSF: correlation with oligoclonal banding and demyelinating disease.

The detection of raised immunoglobulin and the presence of oligoclonal bands (OCBs) on electrophoresis of multiple sclerosis (MS) CSF has been a useful diagnostic test, but a universal antigen to which these MS antibodies are directed has yet to be found. Potentially immunogenic heat shock proteins (HSPs) are preferential expressed in vitro in human oligodendrocytes compared with other glia, and in situ in oligodendrocytes found within the plaques of MS. Immunoreactivity directed against HSPs might therefore contribute to the immune-mediated demyelinating process found in MS. We examined this possibility by quantitatively (ELISA) measuring antibodies directed against a recombinant human HSP (HSP60) in CSF from 18 MS patients, and compared them with eight patients with acute disseminated encephalomyelitis, 12 with demyelinating peripheral neuropathies, and 59 with other neurologic diseases. Immunoblotting was used to confirm the specificity of the antibodies for binding to HSP60. We found a statistically significant correlation between antibody titers to HSP60 and the presence of OCBs in CSF. These results support the notion that HSP expression in the CNS, such as that observed in MS, may be immunogenic, leading to localized HSP antibody secretion. Such HSP-directed immunoreactivity could play a role in the pathogenesis of MS and other immune-mediated disorders of the nervous system.

Antibodies↗
PubMed1992

Production of soluble autocrine inhibitory factors by human glioma cell lines.

Gliomas in vitro exhibit density-limited growth upon the attainment of confluency, an effect usually attributed to cell-cell contact inhibition. Since gliomas have been demonstrated to secrete an array of soluble factors which can enhance tumor growth, we undertook this study to ascertain whether production of soluble factors by the tumor may also inhibit growth in an autocrine manner, and whether production of such factors is associated with the growth phase of the glioma. We observed that cell-conditioned medium (supernatants) from non-confluent glioma cultures induced growth, while confluent culture supernatants produced pronounced growth suppression. These latter supernatants enhanced proliferation of non-transformed astrocytes. Supernatants derived from all stages of confluency produced inhibition of lymphocyte proliferation. To characterize these factors, dialyzed supernatant was tested and found to continue to produce lymphocyte suppression but no glioma growth limitation. Growth of tumors in indomethacin or in acetylsalicylic acid to abolish prostanoid synthesis abrogated the inhibitory influence on glioma growth but only partially reversed the lymphocyte suppressive capacity. These studies suggest that gliomas do produce a growth phase dependent autocrine inhibitory factor(s), and that the production of these small molecular weight factors is at least partially under control of the cyclooxygenase pathway.

Adult↗
PubMed1992

Differential expression of heat shock proteins by human glial cells.

Heat shock proteins (HSP) have been implicated in the interactions between the gamma delta T lymphocyte population and target tissues. gamma delta T cells are found in increased numbers in multiple sclerosis (MS) plaques compared to their proportion in peripheral blood, co-localizing with oligodendrocytes (OGC) expressing HSP. We have demonstrated that such gamma delta T cells can induce in vitro lysis of human adult-derived OGC. Using immunohistochemical and flow cytometry techniques, we examined the constitutive and/or inducible expression of HSP in or on adult human-derived glial cell cultures in vitro. HSP70 was expressed in OGC maintained at basal temperature, but the expression of the inducible HSP70 protein was upregulated by a prior 43 degrees C heat exposure. HSP70 could not be detected within astrocytes (GFAP+ cells), whether heat stress was applied or not. Constitutive expression of HSP60 could be discerned on the surface of all OGC under non-stressed culture conditions. Only some astrocytes demonstrated minor punctate surface HSP60 staining, whereas the remainder did not express HSP60 constitutively. These observations raise the possibility that OGC, by virtue of their differential expression of HSP compared to other glial cells, may be particularly prone to interaction with HSP-reactive gamma delta T cells. Such findings may further implicate gamma delta T cells in the pathogenesis of MS, a putative autoimmune disease in which immune-mediated injury is directed specifically against the oligodendrocyte-myelin unit within the central nervous system.

Adult↗
PubMed1992

Prospective serial analysis of interleukin-2 and soluble interleukin-2 receptor in relapsing-remitting multiple sclerosis.

We performed a longitudinal analysis of serum interleukin-2 (IL-2) and soluble IL-2 (sIL-2R) concentrations in 60 patients with relapsing-remitting (R-R) multiple sclerosis (MS) as well as in 33 age- and sex-matched normal controls. Overall, we found that serum IL-2 levels remained low (less than 10 U/ml) and did not change appreciably over time; however, marked fluctuations in sIL-2R levels were observed in both the patient and control groups. Using patients as their own controls, we calculated an interrelapse (disease stable) mean sIL-2R concentration as a baseline for comparison with relapse values; sIL-2R levels greater than the 90th percentile of the Student's t distribution of stable values were defined as "peaks." There were a total of 27 sIL-2R peaks, eight (30%) of which correlated with clinical relapses but were potentially predictive of only 18% (8/45) of all the recorded clinical relapses. There was no difference in disease severity (Expanded Disability Status Scale) score between peak-correlated and noncorrelated relapses. Our data suggest that despite reports of elevated levels of IL-2 and sIL-2R in MS, neither may be a useful marker for predicting clinical disease activity in R-R MS.

Adolescent↗
PubMed1991

Gamma-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo.

Reactive gliosis is a characteristic response of astrocytes to inflammation and trauma of the central nervous system. To investigate whether soluble factors (cytokines) from inflammatory mononuclear cells that accumulate at lesion sites can provide the cellular signals to initiate gliosis and to identify such cytokines, we have tested and found that supernatants derived from subsets of activated human T lymphocytes (CD8+ or CD4+) are potent mitogens for cultured human adult astrocytes. This effect is blocked by a neutralizing antibody to gamma-interferon (IFN). Recombinant IFN alone can induce proliferation of human adult astrocytes in vitro and increase the extent of trauma-initiated gliosis in the adult mouse brain. The astrocyte proliferation-inducing activity of supernatants of glial cultures treated with IFN can be completely blocked with IFN-neutralizing antibody, suggesting that the proliferative effect does not require intermediary cytokines or cells. These results implicate IFN as an important mediator of the gliosis observed in pathologic conditions of the adult central nervous system associated with infiltrating lymphocytes.

Animals↗
PubMed1991

Differential responses of CD4+CD45RA+ and CD4+CD29+ subsets to activated CD8+ cells: enhanced stimulation of the CD4+CD45RA+ subset by cells from patients with multiple sclerosis.

To examine whether functionally different CD4+ cells respond uniformly to the immunoregulatory influences of allogeneic activated CD8+ cells (*CD8+), we subfractionated the CD4+ population into two subsets, based on the high expression of either CD45RA or CD29. We confirmed that the CD45RA+ cells proliferated poorly in response to soluble anti-CD3 mAb, compared to the vigorous response obtained with the CD29+ subset; the CD45RA+ cells were more responsive to stimulation with Con A. Using normal healthy controls, we found that whereas *CD8+ had a significant suppressive effect on the proliferation of the CD29+ subset, they augmented the mitogen-induced proliferative response of the CD45RA+ cells. We further demonstrated that *CD8+ derived from MS patients augmented the response of the CD45RA+ subset to a significantly higher degree compared to healthy age- and sex-matched controls. There were no significant differences between the degree of suppression exerted by the *CD8+ of either the MS or the control group on the CD29+ cells. These results demonstrate that helper/memory CD4+CD29+ cells are more sensitive to the suppressive influences of *CD8+ compared to the CD4+CD45RA+ subset. In addition, in MS, *CD8+ may contribute to a more pronounced "on" signal for virgin CD4+CD45RA+ cells, which might serve as a means to perpetuate the autoimmune disease process.

Adult↗
PubMed1991

Peripheral blood gamma-delta T cells lyse fresh human brain-derived oligodendrocytes.

T cells are postulated to contribute to the injury of the oligodendrocyte-myelin complex underlying the demyelinating disease multiple sclerosis (MS). The apparent lack of class I or II major histocompatibility complex (MHC) expression in situ on human oligodendrocytes and the consistent failure to identify a universal myelin antigen in MS suggest that the immune damage might be mediated by effector T cells that are capable of reacting in an antigen-nonspecific and possibly MHC-unrestricted manner, such as T cells expressing the gamma-delta T-cell receptor. Since gamma-delta T cells are reported to be present in MS plaques and an increased number are found in the cerebrospinal fluid of patients with MS, we directly examined whether gamma-delta T cells are capable of inducing injury to human oligodendrocytes. We found, using a 6-hour 51Cr release assay, that oligodendrocytes cultured from surgically resected human brain specimens were effectively lysed in a dose-dependent manner by human gamma-delta T cells (28 +/- 5% mean specific lysis, n = 6, at an effector-target ratio of 20:1). Although heat shock protein HSP72, a putative gamma-delta T-cell recognition molecule, could be induced in vitro in our oligodendrocytes, an antibody to HSP72 did not inhibit gamma-delta T cell-mediated lysis of oligodendrocytes. These results suggest that gamma-delta T cells gaining entry into the central nervous system may be deleterious to oligodendrocytes and thus may contribute to the pathogenesis of MS.

Autoimmune Diseases↗
PubMed1991

Phenotypic and functional characteristics of activated CD8+ cells: a CD11b-CD28- subset mediates noncytolytic functional suppression.

Freshly isolated human CD8+ cells can be divided into mutually exclusive subsets bearing the phenotypes CD11b+(CD28-) or CD28+(CD11b-). We found that activation of CD8+ cells with anti-CD3 mAb and IL-2 preferentially expanded the CD11b-(CD28+) subset. This subset, when separated and activated independently, mediated both functional suppression and lectin-dependent cell cytotoxicity (LDCC). CD28- cells, prepared by elimination of the CD 28+ cells from expanded unfractionated CD8+ cell cultures, retained functional suppressor activity but demonstrated reduced LDCC compared to either the CD28+(CD11b-)-enriched fraction or the unfractionated CD8+ population. The majority of the CD28- cells were also CD11b-, reflecting the observation that initially CD11b+ cells lose CD11b expression following activation with anti-CD3 mAb and IL-2. Our results therefore indicate that CD8+ cells deriving from the CD11b+CD28- subset, but expressing neither CD11b nor CD28 after activation, represent the main noncytotoxic functional suppressor cell in the mitogen "activated" suppressor assay. The preferential expansion of CD8+CD28+ cells relative to CD8+CD28- cells, if occurring in vivo in the central nervous system (CNS) compartment, would be consistent with observed phenotypic analysis of cerebrospinal fluid-derived T cells and might contribute to the reduced functional suppressor activity previously found for CNS compared to peripheral blood-derived lymphocytes.

Antibodies, Monoclonal↗
PubMed1990

Immunoregulatory properties of T-cell lines derived from the systemic and intrathecal compartments: a phenotypic and functional study.

To determine whether immune regulation can differ within the intrathecal and systemic compartments, we compared phenotypic markers and functional properties of in vitro anti-CD3 monoclonal antibody-stimulated, interleukin 2-expanded lymphoid cell lines simultaneously derived from peripheral blood and cerebrospinal fluid of individual donors (n = 9). We found that the proportions of total CD8+ T cells and of the putative CD8+ suppressor effector subset (CD28-) were lower in the cell lines derived from cerebrospinal fluid compared with cultures derived from peripheral blood (p less than 0.025 and p less than 0.005, respectively; paired t test), whereas the total CD4+ T-cell proportion was higher (p less than 0.025). For a donor subgroup with "normal" peripheral blood cell-mediated activated suppressor function (63 +/- 2%), mean suppressor cell function mediated by unfractionated or CD8(+)-enriched cells derived from cerebrospinal fluid was significantly lower (38 +/- 7%; p less than 0.01, paired t test). For a donor subgroup with "low" peripheral blood cell-mediated suppression (-1 +/- 10%), suppression mediated by cerebrospinal fluid cells was also "low" (9 +/- 12%). Our results support the postulate that the immune response may be differentially regulated between the central nervous system and peripheral blood compartments.

Adult↗
PubMed1990

Human oligodendrocytes are susceptible to cytolysis by major histocompatibility complex class I-restricted lymphocytes.

The majority of human oligodendrocytes in enriched glial cell cultures expresses class I major histocompatibility complex (MHC) antigens. We used a 51Cr release assay to study the susceptibility of oligodendrocyte-enriched glial cells to MHC-restricted and non-restricted immune-mediated cytolysis. Mitogen-activated mononuclear cells induced significant lysis in a lectin-dependent cytotoxicity assay. Mononuclear cells allo-activated in a one-way mixed lymphocyte culture with E- cells from the glial cell donor induced a significantly higher degree of oligodendrocyte cytolysis than mononuclear cells activated with E- cells bearing MHC-class I antigens discordant with the glia. Cytolysis by alloactivated unfractionated lymphocytes and by purified CD8+ lymphocytes was reduced by an anti-class I antibody (W6/32). Our findings suggest that human oligodendrocytes can be susceptible targets for MHC class I-restricted lysis.

CD4-Positive T-Lymphocytes↗
PubMed1989

Activated suppressor cell function in severely disabled patients with multiple sclerosis.

Defective suppressor cell function has previously been demonstrated in patients with multiple sclerosis (MS) with progressive disease and moderate degrees of disability. In the present study activated suppressor cell function was assessed in patients with documented progressive disease who, at the time of study, had experienced severe disability (Kurtzke score greater than or equal to 6.5) for at least 2 years. We found that mean suppressor levels were significantly increased in this patient group compared with the suppressor levels in the MS patient group with progressive disease but only moderate disability (Kurtzke score of less than or equal to 6.0 within 2 years of study) (59 +/- 8% vs 19 +/- 7%, respectively, p less than 0.01). The mean value in the latter group was significantly reduced compared with the mean value for normal control subjects (47 +/- 4%, p less than 0.01), a finding consistent with previous reports. The results of this study indicate that suppressor cell function, as measured by our assay system, need not be defective in MS patients who have become severely disabled from the progressive form of the disease. Whether the patients who are now severely disabled from progressive MS passed through a phase of disease associated with the same suppressor defects as found in the progressive patients currently with moderate disability will remain speculative until long-term longitudinal studies are performed.

Adult↗