Prolactin in combination with interferon-ß reduces disease severity in an animal model of multiple sclerosis.

Previous work has demonstrated that the hormone prolactin promotes oligodendrocyte precursor proliferation and remyelination following lysolecithin-induced demyelination of the mouse spinal cord. Prolactin, however, can elicit pro-inflammatory responses, and its use in the prototypical demyelinating and inflammatory condition, multiple sclerosis (MS), should thus be approached cautiously. Here, we sought to determine whether recombinant prolactin could alter the course of experimental autoimmune encephalomyelitis (EAE), an inflammatory animal model of MS. Consistent with previous literature, we found that prolactin activated leukocytes in vitro. Daily treatment with prolactin from around the time of onset of clinical signs, for 9 (days 9 to 17) or 25 (days 9 to 33) days did not increase clinical or histological signs of EAE over that of vehicle-treated mice. Instead, the combination of prolactin and a suboptimal dose of recombinant murine interferon-ß resulted in (days 9 to 17 group) or trended towards (days 9 to 33 group), a greater amelioration of clinical signs of EAE, compared to either treatment alone or to vehicle controls. Histological analyses corroborated the clinical EAE data. These results suggest that prolactin may be beneficial when administered in combination with interferon-ß in MS.

Dual effects of daily FTY720 on human astrocytes in vitro: relevance for neuroinflammation.

BACKGROUND: FTY720 (fingolimod, Gilenya) is a daily oral therapy for multiple sclerosis that readily accesses the central nervous system (CNS). FTY720 is a structural analog to the sphingolipid sphingosine-1-phosphate (S1P) and is a cognate ligand for the S1P G-protein coupled receptors (S1PR). Studies in experimental autoimmune encephalomyelitis using mice with conditionally deleted S1P1R from astrocytes indicate that one beneficial effect of FTY720 in this model is via downregulating external receptors, which inhibits responses induced by the natural ligand. Another proposed effect of FTY720 on neuroinflammation is its ability to maintain persistent signaling in cells via internalized S1P1R resulting in functional responses that include suppressing intracellular calcium release. We used human fetal astrocytes to investigate potential dual inhibitory- and function-inducing effects of daily FTY720 on responses relevant to neuroinflammation. For the inhibitory effects, we used signaling and proliferation induced by the natural ligand S1P. For the function-inducing responses, we measured inhibition of intracellular calcium release stimulated by the proinflammatory cytokine, interleukin (IL)-1ß. METHODS: Astrocytes derived from human fetal CNS specimens and maintained in dissociated cultures were exposed to 100 nM of the biologically active form of FTY720 over a dosing regimen that ranged from a single exposure (with or without washout after 1 h) to daily exposures up to 5 days. Responses measured include: phosphorylation of extracellular-signal-regulated kinases (pERK1/2) by Western blotting, Ki-67 immunolabeling for cell proliferation, IL-1ß-induced calcium release by ratiometric fluorescence, and cytokine/chemokine (IL-6, CXCL10) secretions by ELISA. RESULTS: We observed that a single addition of FTY720 inhibited subsequent S1PR ligand-induced pERK1/2 signaling for >24 h. Daily FTY720 treatments (3-5 days) maintained this effect together with a loss of proliferative responses to the natural ligand S1P. Repeated FTY720 dosing concurrently maintained a functional cell response as measured by the inhibition of intracellular calcium release when stimulated by the cytokine IL-1ß. Recurrent FTY720 treatments did not inhibit serum- or IL-1ß-induced pERK1/2. The secretions of IL-6 and CXCL10 in response to IL-1ß were unaffected by FTY720 treatment(s). CONCLUSION: Our results indicate that daily FTY720 exposures may regulate specific neuroinflammatory responses by desensitizing astrocytes to external S1PR stimuli while sustaining cellular influences that are independent of new surface S1PR activation.

Reduction of the peripheral blood CD56(bright) NK lymphocyte subset in FTY720-treated multiple sclerosis patients.

FTY720 (fingolimod) treatment of multiple sclerosis (MS) results in lymphopenia due to increased recruitment into and decreased egress from secondary lymphoid organs of CCR7(+) lymphocytes. Although absolute numbers of NK lymphocytes were reported as being unaltered in FTY720-treated MS patients (MS-FTY), such analyses did not detect a change in a minor subset. Because expression of CCR7 has been described on CD56(bright) NK cells, a minority population of NK cells, we investigated the effect of FTY720 treatment on the phenotype and function of human NK cells in the peripheral circulation of MS patients. MS-FTY patients displayed a decreased proportion of peripheral CD56(bright)CD62L(+)CCR7(+) NK cells compared with untreated MS and healthy donors. In vitro treatment with FTY720-P increased migration of untreated donor NK cells to CXCL12 while reducing the response to CX3CL1 with similar migration responses seen in NK cells from MS-FTY patients. FTY720-P inhibited sphingosine 1-phosphate-directed migration of CD56(bright) and CD56(dim) NK cells subsets from untreated healthy donors. IL-12- and IL-15-stimulated NK cells from MS-FTY patients displayed similar capacity to produce IFN-γ, TNF, IL-10, and MIP-1α cytokines/chemokines compared with NK cells from untreated healthy donors and displayed comparable levels of degranulation in response to K562 tumor cells compared with untreated donors. Subset alterations and function of NK cell populations will need to be considered as part of assessing overall immunosurveillance capacity of patients with MS who will receive sustained FTY720 therapy.

Comparison of polarization properties of human adult microglia and blood-derived macrophages.

Both microglia, the resident myeloid cells of the CNS parenchyma, and infiltrating blood-derived macrophages participate in inflammatory responses in the CNS. Macrophages can be polarized into M1 and M2 phenotypes, which have been linked to functional properties including production of inflammation association molecules and phagocytic activity. We compare phenotypic and functional properties of microglia derived from the adult human CNS with macrophages derived from peripheral blood monocytes in response to M1 and M2 polarizing conditions. Under M1 conditions, microglia and macrophages upregulate expression of CCR7 and CD80. M2 treatment of microglia-induced expression of CD209 but not additional markers CD23, CD163, and CD206 expressed by M2 macrophages. M1-polarizing conditions induced production of IL-12p40 by both microglia and macrophages; microglia produced higher levels of IL-10 under M1 conditions than did macrophages. Under M2 conditions, microglia ± LPS produced comparable levels of IL-10 under M1 conditions whereas IL-10 was induced by LPS in M2 macrophages. Myelin phagocytosis was greater in microglia than macrophages under all conditions; for both cell types, activity was higher for M2 cells. Our findings delineate distinctive properties of microglia compared with exogenous myeloid cells in response to signals derived from an inflammatory environment in the CNS.

Reconstitution of circulating lymphocyte counts in FTY720-treated MS patients.

FTY720 (Fingolimod) reduces multiple sclerosis disease activity by inducing lymphopenia and inhibiting lymphocyte re-entry from lymph nodes. Peripheral lymphocyte reconstitution following drug discontinuation has been considered relatively rapid (2-4 weeks), based on short-term studies. We investigated the kinetics of lymphocyte reconstitution in MS patients in open label extension phases of FTY720 clinical trials who discontinued therapy after prolonged use (>1-5 years), and examined histological features of a mediastinal lymph node obtained from a lymphopenic FTY720 patient. Although three patients showed reconstitution of peripheral lymphocytes within the predicted timeline, two patients continued to be lymphopenic 9 and 34 months after therapy cessation. Lymph nodes from the latter patient showed preserved architecture. Notwithstanding preserved lymph node integrity, time for lymphocyte reconstitution after prolonged FTY720 therapy can be significantly greater than predicted by shorter-term studies. This is relevant for clinical decisions regarding management of patients using this therapy and for introducing alternate therapies.

Absence of the cellular prion protein exacerbates and prolongs neuroinflammation in experimental autoimmune encephalomyelitis.

Although the physiological roles of the cellular prion protein (PrP C) remain to be fully elucidated, PrP C has been proposed to represent a potential regulator of cellular immunity. To test this hypothesis, we evaluated the consequences of PrP C deficiency on the course of experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein peptide. Consistent with augmented proliferative responses and increased cytokine gene expression by myelin oligodendrocyte glycoprotein-primed Prnp-/- T cells, PrP C-deficient mice demonstrated more aggressive disease onset and a lack of clinical improvement during the chronic phase of experimental autoimmune encephalomyelitis. Acutely, Prnp-/- spinal cord, cerebellum, and forebrain exhibited higher levels of leukocytic infiltrates and pro-inflammatory cytokine gene expression, as well as increased spinal cord myelin basic protein and axonal loss. During the chronic phase, a remarkable persistence of leukocytic infiltrates was present in the forebrain and cerebellum, accompanied by an increase in interferon-gamma and interleukin-17 transcripts. Attenuation of T cell-dependent neuroinflammation thus represents a potential novel function of PrP C.

Secretory products of multiple sclerosis B cells are cytotoxic to oligodendroglia in vitro.

B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-ß1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.

Differential responses of human microglia and blood-derived myeloid cells to FTY720.

Human microglia, monocyte-derived dendritic cells (DCs) and macrophages ex vivo express relatively higher levels of sphingosine-1-phosphate (S1P) receptor 1 (S1P1) mRNA as compared to other receptor subtypes. The S1P agonist FTY720 decreased ERK phosphorylation and induced myosin light chain (MLC) II phosphorylation only in macrophages and DCs. FTY720 inhibited IL-12p70 production (CD40L induced) by DCs and macrophages but not microglia (poly I:C induced). IL-10 production was increased in DCs and unaffected in other myeloid cells. Despite similar receptor expression patterns, the distinct myeloid cell populations present in the human CNS, under steady-state or inflammatory conditions, exhibit differential responses to FTY720.

Exploring the roles of CD8(+) T lymphocytes in the pathogenesis of autoimmune demyelination.

Multiple sclerosis (MS) is an autoimmune disease characterized by mononuclear cell infiltrates, focal demyelination, and the development of sclerotic plaques within the central nervous system. Although CD8(+) T lymphocytes are more abundant than CD4(+) T lymphocytes in MS lesions, the latter cell type has been most commonly implicated in the genesis of this disease. Recent evidence, however, suggests that both T cell populations and their various subsets are able to contribute to disease initiation and progression. To gain insight into disease mechanisms of potential relevance to MS, a variety of animal models have been developed. Foremost among these has been experimental autoimmune encephalomyelitis (EAE), a rodent model of MS induced by the immunization of genetically susceptible animals with peptides derived from myelin-associated proteins. While EAE has contributed greatly to our understanding of mechanisms involved in autoimmune demyelination, this model has been of limited use as far as shedding light on the possible contributions of CD8(+) T lymphocytes to disease pathogenesis. Herein, we review evidence supporting a role for CD8(+) T lymphocytes in both MS and EAE and also highlight several novel murine systems designed for investigating the role(s) of CD8(+) T cells in autoimmune demyelination.

Distinct properties of circulating CD8+ T cells in FTY720-treated patients with multiple sclerosis.

OBJECTIVE: To define the capacity of peripheral blood CD8(+) T cells from patients with multiple sclerosis (MS) receiving fingolimod (FTY720) to migrate in response to chemokines that contribute to trafficking into the central nervous system. DESIGN: Peripheral blood T cells of FTY720-treated patients with MS (MS-FTY) are mainly CD8(+) CCR7⁻ effector memory cells as CCR7(+) T cells are inhibited from exiting from secondary lymph nodes. Migration of CD8(+) T cells from MS-FTY patients and untreated donors to chemokines CXCL12 and CCL2 was assayed in vitro. Expression of CCL2 receptor (CCR2), CCR7, CD28, and CD27 on CD8(+) T cells was determined by flow cytometry. SETTING: Montreal Neurological Institute's clinical research unit. Patients The MS-FTY patients were part of the extension phase of FTY720 clinical trials for relapsing-remitting MS. RESULTS: In vitro addition of active (phosphorylated) FTY720 increased migration of CD8(+) T cells from untreated patients to CXCL12 and CCL2. The CD8(+) or CD8(+) CCR7⁻ T cells from MS-FTY patients migrated less to CXCL12 and CCL2 compared with those from untreated donors. The proportion of CD8(+) CCR7⁻ cells that express the CCL2 chemokine receptor, CCR2, was significantly reduced in the MS-FTY group. The CD8(+) CCR7⁻ cells from the MS-FTY patients were enriched with CD27⁻ CD28⁻ (late effector) memory cells, a population with reduced expression of CCR2 compared with early (CD27(+) CD28(+)) effector memory cells. CONCLUSIONS: Therapy with FTY720 results in a subset of CD8(+) T cells with distinct functional migratory properties dominating the peripheral circulation. The expected forthcoming use of FTY720 as a sustained therapy for MS will clarify how this redistribution of lymphocyte populations affects the overall process of immune surveillance.

Antigen-induced Pten gene deletion in T cells exacerbates neuropathology in experimental autoimmune encephalomyelitis.

The Pten tumor suppressor gene is critical for normal intrathymic development of T cells; however, its role in mature antigen-activated T cells is less well defined. A genetically crossed mouse line, Pten(fl/fl) GBC, in which Pten gene deletions could be primarily confined to antigen-activated CD8+ T cells, enabled us to evaluate the consequences of Pten loss on the course of experimental autoimmune encephalomyelitis. Compared with Pten(fl/fl) controls, myelin oligodendrocyte glycoprotein (MOG) peptide-immunized Pten(fl/fl) GBC mice developed more severe and protracted disease. This was accompanied by increased spinal cord white matter myelin basic protein depletion and axonal damage, as well as a striking persistence of macrophage and granzyme B-expressing cellular neuroinfiltrates in the chronic phase of the disease. This persistence may be explained by the observation that anti-CD3 activated Pten(fl/fl) GBC T cells were more resistant to proapoptotic stimuli. Consistent with the predicted consequences of Pten loss, purified CD8+ T cells from Pten(fl/fl) GBC mice displayed augmented proliferative responses to anti-T-cell receptor stimulation, and MOG-primed Pten(fl/fl) GBC T cells exhibited a reduced activation threshold to MOG peptide. Pten(fl/fl) GBC mice also developed atypical central nervous system disease, manifested by prominent cervical cord and forebrain involvement. Collectively, our findings indicate that the phosphatidylinositol 3-kinase signaling pathway is an essential regulator of CD8+ T-cell effector function in experimental autoimmune encephalomyelitis.