Asim Diab

High IL-6 and low IL-10 in the central nervous system are associated with protracted relapsing EAE in DA rats

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated, inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS). The mechanisms behind differences in clinical course of EAE in different rat strains have not been defined. We induced acute EAE in Lewis rats and protracted relapsing EAE (PR-EAE) in DA rats and examined mRNA expression of IL-1 beta, IL-6, IL-10, IL-12, and TNF-beta in brain tissue sections, cerebrospinal fluid (CSF) cells, and lymph node cells. IL-1 beta, IL-12 and TNF-beta mRNA expression in brain tissue sections appeared early and peaked at the height of clinical signs in both acute and PR-EAE, consistent with a disease-promoting role for these cytokines. High levels of IL-6 mRNA-expressing cells were present in CNS and lymph node cells in PR-EAE, while almost absent in acute EAE. In contrast, IL-10 was very low in PR-EAE but strongly expressed in acute EAE, in particular during clinical recovery. Regulatory changes of IL-6 and IL-10 both systemically and within the CNS, but with temporal differences between compartments, seem pivotal for development of PR-EAE in DA rats. These findings could have relevance for pathogenesis and treatment of MS.

Haemophilus influenzae and Streptococcus pneumoniae induce different intracerebral mRNA cytokine patterns during the course of experimental bacterial meningitis

Using in situ hybridization with radiolabelled oligonucleotide probes, we studied the mRNA expression of IL‐1β, IL‐4, IL‐6, IL‐10, IL‐12, tumour necrosis factor‐alpha (TNF‐α), TNF‐β, interferon‐gamma (IFN‐γ), and transforming growth factor‐beta (TGF‐β) in the brain during the lethal course of experimental meningitis in a rat model inoculated intracisternally with Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae and in uninfected control rats inoculated with the same volume of PBS. The production of IL‐1β, IL‐4, IL‐6 and IFN‐γ was also evaluated by immunohistochemistry. In the brain of Hib‐inoculated rats, there was marked mRNA expression of IL‐1β, IL‐6, TNF‐α, IL‐12 and IFN‐γ. IL‐1β, IL‐6 and TNF‐α were up‐regulated throughout the observation period at 2, 8 and 18 h post‐inoculation (p.i.), with similar patterns of induction. The Th1 cytokines IFN‐γ and TNF‐β were up‐regulated within 8 h p.i. IL‐10 and TGF‐β were down‐regulated at 18 h p.i., while IL‐4 was not detected. In contrast, the brain of S. pneumoniae‐inoculated rats showed lower levels of IL‐1β, IL‐6 and TNF‐α, but higher levels of TNF‐β and detectable mRNA expression of IL‐4 when compared with Hib‐inoculated rats. IL‐12, IFN‐γ, IL‐10 and TGF‐β exhibited similar patterns of induction in the brains of Hib‐ and S. pneumoniae‐inoculated rats. At 18 h p.i., immunohistochemistry showed similar patterns of IL‐1β, IL‐4, IL‐6 and IFN‐γ as mRNA expression in the brains of Hib‐ and S. pneumoniae‐inoculated rats. The differences of cytokine profiles induced by the two bacterial strains may imply that different immunomodulating approaches should be considered, depending on etiology.

Linomide suppresses acute experimental autoimmune encephalomyelitis in Lewis rats by counter-acting the imbalance of pro-inflammatory versus anti-inflammatory cytokines

Linomide (quinoline-3-carboxamide) is a synthetic immunomodulator that suppresses several experimental autoimmune diseases. Here we report the effects of Linomide on experimental autoimmune encephalomyelitis (EAE), a CD4+ T cell-mediated animal model of multiple sclerosis (MS). EAE induced in Lewis rats by inoculation with homogenized guinea pig spinal cord and Freunds complete adjuvant was strongly suppressed by Linomide administered daily subcutaneously from the day of inoculation. Linomide dose-dependently delayed the interval between immunization and onset of clinical EAE, and reduced severity of EAE symptoms. These clinical effects were associated with dose-dependent down-modulation of myelin antigens-induced T cell responses and by suppression of the proinflammatory cytokines IFN-gamma and TNF-alpha, and upregulation IL-4, IL-10 and TGF-beta as evaluated by in situ hybridization for mRNA expression in spleen mononuclear cells and spinal cord sections. These findings suggest that Linomide could be useful in certain T cell dependent autoimmune diseases.

Astrocytes induce hyporesponses of myelin basic protein-reactive T and B cell function

We have isolated infiltrating mononuclear cells (inMNC) and lymph node MNC (lnMNC) from Lewis rats with actively induced experimental allergic encephalomyelitis (EAE), and compared their responses to myelin basic protein (MBP). MBP-induced proliferation and MBP-specific IgG secreting cells were lower in inMNC compared to lnMNC, while MBP-reactive IFN-gamma secreting cells in inMNC were higher. Using an in vitro culture system, we observed that astrocytes derived from newborn Lewis rats not only suppressed T cell proliferation and IFN-gamma production but also reduced MBP-specific IgG production by B cells. Astrocyte-derived soluble factor (s), rather than direct cell-to-cell interactions, seems to be responsible for the inhibitory effect. Supernatants from IFN-gamma-stimulated astrocytes exhibited stronger suppressive effects than supernatants from unstimulated astrocytes, whereas supernatants from microglia did not cause downregulation of T and B cell functions. These results indicate that astrocytes are major effector cells in inhibiting functions of MBP-reactive T and B cells. Astrocytes down-regulated expression of ICAM-1 and MHC class II in lnMNC, but did not induce apoptosis of lnMNC. The results in the present study do not exclude the possibility that astrocytes induce T cell apoptosis in a cognate fashion. Taken together, the inhibitory properties of astrocytes may contribute to the confinement of inflammatory lesions in multiple sclerosis and EAE. Our report compares immunoreactivities of inMNC and lnMNC in EAE and elucidates the role of astrocytes in the inactivation of MBP-reactive T and B cells.

Augmented levels of macrophage and Th1 cell-related cytokine mRNA in submandibular glands of MRL/lpr mice with autoimmune sialoadenitis

MRL/Mp‐lpr/lpr (MRL/lpr) mice spontaneously develop destructive inflammation of the salivary and lachrymal glands resembling Sjögrens syndrome (SS), representing an animal model to study this disease. We used in situ hybridization with synthetic radiolabelled oligonucleotide probes to examine expression of mRNA encoding pro‐ and anti‐inflammatory cytokines in submandibular glands of 2, 3, 4 and 5‐month‐old MRL/lpr mice. Phenotypic composition of submandibular gland infiltrates was evaluated by immunohistochemistry. Cells expressing tumour necrosis factor‐alpha (TNF‐α), IL‐1β, IL‐6 and IL‐12 mRNA were strongly up‐regulated at about the time of onset of sialoadenitis, suggesting a role of these cytokines in development of the disease. Interferon‐gamma (IFN‐γ) and cytolysin mRNA‐expressing cells were gradually up‐regulated over the disease course up to 5 months of age, the time when sialoadenitis is at its height, favouring a role of these cytokines in progression of the disease as well. Low levels of IL‐10 and transforming growth factor‐beta (TGF‐β) mRNA‐expressing cells were observed at 2, 3 and 4 months of age, and were almost undetectable at 5 months. Maximum levels of CD4+, CD8+ and interdigitating/dendritic cells, as well as of MHC class II and MHC class I expression were seen at 3 months, with CD4+ outnumbering CD8+ cells. Maximum levels of macrophages were seen at 4 months of age. These data argue for a major role of the proinflammatory cytokines TNF‐α, IL‐1β, IL‐6, IL‐12, IFN‐γ and cytolysin in initiation and perpetuation of autoimmune sialoadenitis in MRL/lpr mice, probably in conjunction with an insufficiency of the anti‐inflammatory cytokines TGF‐β and IL‐10.

Suppression of macrophage activation with CNI-1493 increases survival in infant rats with systemic Haemophilus influenzae infection

ABSTRACT CNI-1493, a potent macrophage deactivator, was used to treat infant rats systemically infected with Haemophilus influenzae type b (Hib). CNI-1493 was injected 1 h prior to bacterial inoculation and 24 h later and resulted in a 75 percent increased rate of survival compared to that for untreated controls. The effect of CNI-1493 on the inflammatory response was studied by immunohistochemical detection of individual tumor necrosis factor alpha (TNF-α)-, interleukin 1 beta (IL-1β)-, and gamma interferon (IFN-γ)-producing cells in the spleen. A significant reduction of the incidence of TNF-α- and IL-1β-expressing cells was found for CNI-1493-treated animals. IFN-γ expression was not suppressed by CNI-1493, indicating that cytokine inhibition was specific in macrophages. CNI-1493 significantly reduced the number of infiltrating granulocytes in the brain from that for controls. This study provides evidence that CNI-1493 protects against lethal Hib infection by deactivating the inflammatory cascade in infant rats.

Cytokine and chemokine mRNA expressing cells in muscle tissues of experimental autoimmune myasthenia gravis

In-situ hybridization with labeled oligonucleotide probes was applied to explore cytokine and chemokine mRNA expression in sections of striated muscle, the target organ in experimental autoimmune myasthenia gravis (EAMG), induced in Lewis rats by immunization with acetylcholine receptor (AChR) and complete Freunds adjuvant (CFA). A transient burst of TNF-alpha, IL-1beta and IL-6 mRNA expressing cells was detected during the early phase of EAMG. This cytokine pattern was related to muscular infiltration of macrophages. Levels of IL-4, IL-10, IFN-gamma, cytolysin and TGF-beta mRNA expressing cells were low and observed mainly during the early phase of EAMG. C-C chemokine RANTES, MCP, MIP-1alpha and MIP-2 mRNA expressing cells were not detected over the course of EAMG. The low and transient expression of cytokines in EAMG muscle tissues suggests that the immune effector responses are unlikely operated by infiltrating cells in muscle. Muscular infiltrations in EAMG are unlikely due to local accumulation of C-C chemokines.

Linomide suppresses chronic-relapsing experimental autoimmune encephalomyelitis in DA rats

Linomide (quinoline-3-carboxamide) is a synthetic immunomodulator that suppresses several experimental autoimmune diseases. Here we report the effects of Linomide on chronic progressive and/or relapsing experimental autoimmune encephalomyelitis (PR-EAE), a CD4+ T cell mediated animal model of multiple sclerosis (MS). PR-EAE induced in DA rats by inoculation with homogenized guinea pig spinal cord and Freunds complete adjuvant, was strongly suppressed by Linomide administered daily subcutaneously from the day of inoculation. Linomide dose-dependently delayed the interval between immunization and onset of clinical PR-EAE, reduced severity and relapse of clinical PR-EAE, and shortened clinical PR-EAE. These clinical effects were associated with the down-modulation of CNS antigen-induced T cell responses and production of proinflammatory cytokines (IFN-gamma and TNF-alpha) as well as with upregulation of IL-4 (except in spleen MNC), IL-10 and TGF-beta in both spleen MNC and the spinal cord. These effects indicate that Linomide can suppress PR-EAE and may mediate its suppressive effects by regulation of cytokines.

Treatment with P2 protein peptide 57–81 by nasal route is effective in Lewis rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated autoimmune disorder of the peripheral nervous system (PNS) that can be actively induced in susceptible animal species and strains by active immunization with PNS myelin + Freunds complete adjuvant (FCA). EAN represents an animal model for studying the immunopathogenesis and treatment of Guillain-Barré syndrome (GBS), which is a major inflammatory demyelinating disease of the PNS in humans. Here, we report that treatment by nasal administration of the neuritogenic peptide 57-81 of the PNS myelin component, P2 protein, dose-dependently suppressed EAN severity and shortened clinical EAN. Clinical EAN relapse induced by rechallenge with BPM + FCA was also prevented in EAN rats receiving high dose P2 peptide. P2 peptide induced suppression of EAN was associated with PNS antigen specific T cell hyporesponsiveness reflected by lymphocyte proliferation, numbers of PNS antigen-reactive IFN-gamma secreting and IFN-gamma mRNA expressing lymph node cells, but elevated levels of PNS antigen reactive TGF-beta mRNA secreting cells. Reduced CD4+ T cell and macrophage infiltrations within the PNS were also observed. Based on these observations, nasal autoantigen administration should be further evaluated, considering its possible future use in GBS.

Prevention of Experimental Autoimmune Neuritis by Nasal Administration of P2 Protein Peptide 57–81

Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-BarnS syndrome (GBS) in humans. Both EAN and GBS are associated with upregulated T and B cells responses to PNS myelin proteins including P2 protein, and by changes of the Thl/Th2 cell balance in favor of Thl. Here we report that EAN can be prevented by the dominant neuritogenic peptide 57-81 of the PNS P2 protein when given nasally before immunization of Lewis rats with bovine PNS myelin (BPM) + Freunds complete adjuvant (FCA). P2 peptide-tolerized rats were also resistant to EAN relapse after challenge with BPM. Tolerance to EAN in rats receiving high dose (60µg/day/rat) P2 peptide nasally was associated with specific T and B cell anergy. This was characterized by the failure of T cells to proliferate in response to PNS myelin antigens, while responsiveness to phytohemagglutinin was retained. Numbers of BPM- and P2 peptide-reactive interferon- γ mRNA expressing lymph node cells were reduced, while levels of P2 peptide-reactive interleukin 4 and transforming growth factor-β mRNA-expressing cells were markedly upregulated on day 18 post immunization in the rats receiving high dose P2 peptide nasally. Tolerance to EAN was also associated with lower CD4+ cell infiltration, low-grade inflammation, or the absence of histological evidence of EAN, as well as with low IL-2 receptor and MHC class II molecule expression within the PNS. This is the first study showing that mucosal tolerance is applicable to EAN and, as an extension, could be considered in GBS.