Type I interferons as immunoregulatory molecules; implications for therapy in experimental autoimmune uveoretinitis

Clinical trials have shown that the type I interferon (IFN)-alpha/beta have some beneficial effects on organ-specific autoimmune diseases, such as Behcet's diseases and multiple sclerosis, although the precise mechanisms remain largely unresolved. T helper cells 1 (Th1)-mediated autoimmune responses are involved in the initiation and/or progression of human uveitis, such as Behcet's disease. The animal model of experimental autoimmune uveoretinitis (EAU), characterized by a monophasic clinical course, has contributed to the understanding of the pathogenesis of human uveitis. Th1 producing IFN-gamma induce EAU development, while Th2 producing IL-4/IL-10 prevent the disease. However, depending on the cytokine milieu, the pro-inflammatory cytokine IFN-gamma may attenuate the autoimmune responses and anti-inflammatory cytokine IL-4 exacerbates it. Chemokines also play a crucial role in EAU development, which might be resolved by Th2-mediated immune responses. The administration of IFN-alpha/beta prevents EAU development, accompanied by a diminished production of IFN-gamma/IL-10. Interestingly, however, IFN-alpha/beta also have some beneficial effects on patients with Th2-like phenotype in addition to Th1-like phenotypes. Thus, the immuno-modulatory action of IFN-alpha/beta may be dependent on the context of cytokine combination and/or their concentrations.     

Cytokines and apoptotic molecules in experimental melanin-protein induced uveitis (EMIU) and experimental autoimmune uveoretinitis (EAU).

The cytokine profile and occurrence of apoptosis during experimental melanin-protein induced uveitis (EMIU) were investigated and compared with that of experimental autoimmune uveoretinitis (EAU). EMIU or EAU was induced in Lewis rats. Eyes were collected at different time points after immunization. Cytokine mRNA expression was identified in the inflammatory cells in the uvea of EMIU rats; IL-2, IFN-gamma and IL-12 increased at the peak of the inflammation, and then tapered off as inflammation subsided. IL-4 and IL-10 increased at the peak of ocular inflammation, and persisted with inflammation resolved. Fas and FasL were expressed consistently in ocular resident cells of EMIU, but were elevated in EAU. In EAU, Bcl-2 expression showed a sharp peak in inflammatory cells but not in the resident cells. In EMIU, high levels of Bcl-2 were present and persisted in both ocular resident and inflammatory cells. Expression of Bax was relatively stable in both EAU and EMIU. Cellular DNA fragmentation was detected in the retinal glial cells of EAU and some inflammatory cells of EMIU. In EMIU, the dynamics of Th1 cytokines were consistent with the ocular inflammation, whereas persistent expression of Th2 cytokines was consistent with their known regulatory role. The continuous high expression of Bcl-2 and the high ratio of Bcl-2 to Bax in the eyes of EMIU may possibly contribute to prevention of ocular tissue damage, and of inflammatory cells from undergoing apoptosis, thus resulting in chronic recurrent inflammation.     

Autoimmunity through cytokine-induced dendritic cell activation

We propose a model where autoimmunity can be viewed as a dynamic system driven by opposite vectors IFN-alpha/beta and TNF. These cytokines drive differentiation of distinct types of DCs, TNF-DCs, or IFN-DCs, which present different antigens leading to distinct autoimmune responses. When balanced, both cytokines synergize in protective immunity. When one of the cytokines prevails, autoimmunity occurs, Type I interferons (IFN-alpha/beta) playing a major role in systemic lupus erythematosus (SLE) and TNF playing a major role in rheumatoid arthritis. This model complements the Type 1/Type 2 paradigm. Therefore, immunity can be viewed as a dynamic system driven by two sets of opposite vectors: IFN-alpha/beta/TNF and IFN-gamma/IL-4.     

Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene

Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL-10 adenovirus (Ad-vIL-10)-mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S-antigen (S-Ag). B10-A mice that received a single unilateral injection of Ad-vIL-10 in the retro-orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL-10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN-gamma and IL-2 were found in cellular supernatants from IRBP-stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL-10 was neutralized completely by injection of a monoclonal anti-vIL-10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL-10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad-vIL-10 was performed in Lewis rats simultaneously with S-antigen in the footpads. This injection determined in situ vIL-10 expression with very low circulating vIL-10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad-mediated gene transfer resulting in systemic and local expression of vIL-10 provide a promising approach for the treatment of uveitis.     

IFN-alpha and IL-18 exert opposite regulatory effects on the IL-12 receptor expression and IL-12-induced IFN-gamma production in mouse macrophages: novel pathways in the regulation of the inflammatory response of macrophages

We characterized the IL-12 response of mouse macrophages in terms of modulation of IFN-gamma production by cytokines (IFN-alpha and IL-18) and regulation of IL-12 receptor expression. Beta1 and beta2 IL-12R chain mRNA expression increased with time in culture in the absence of exogenous stimulation, with concomitant acquisition of responsiveness to IL-12 for IFN-gamma production. Expression of the IL-12R beta1 chain mRNA was increased further following IL-12 treatment as a consequence of IFN-gamma expression. IL-12 response was regulated differentially by IFN-alpha and IL-18. Neutralization of endogenous type I IFN increased IFN-gamma secretion, whereas exogenous IFN-alpha reduced it. In contrast, IL-18 enhanced IFN-gamma mRNA accumulation and IFN-gamma secretion in IL-12-stimulated, but not -untreated, cultures. The opposite effects exerted by IFN-alpha and IL-18 mirror their mutual capacity of regulating-in a negative or positive manner, respectively-the expression of the IL-12R beta1 chain. We suggest that differential regulation of IL-12 response by IFN-alpha and IL-18 can represent previously unrecognized regulatory mechanisms for maintaining suitable levels of differentiation/activation in macrophages.     

Role of Th1 and Th2 cytokines in immune response to uncomplicated Plasmodium falciparum malaria

The relative balance between Th1 and Th2 cytokines appears crucial, since the role of cytokines has been evaluated in several studies by comparison of clinically heterogeneous groups of patients. The aim of this study is to determine the role of proinflammatory Th1 cytokines, interleukin-12 (IL-12) and gamma interferon (IFN-gamma), and anti-inflammatory Th2 cytokines, IL-4 and IL-10, in a homogeneous group of patients with uncomplicated Plasmodium falciparum malaria. Levels of IL-12, IFN-gamma, Il-4, and IL-10 in serum for 20 adult patients and 15 healthy control subjects were determined by an immunoenzymatic assay. Serum levels of Th1 cytokines, IL-12 (8.6 +/- 2.8 pg/ml; controls, 3.2 +/- 0.7 pg/ml) and IFN-gamma (39.2 +/- 67.6 pg/ml; controls, 8.4 +/- 6.3 pg/ml), were significantly increased at admission; 3 days later, levels of IL-12 in serum remained significantly high (8.8 +/- 2.6 pg/ml), whereas IFN-gamma levels returned to control values. The anti-inflammatory response of Th2 cytokines (IL-10 and IL-4) was distinct. Levels of IL-10 in serum were not significantly increased at day 0 and day 3 (306.6 +/- 200.4 pg/ml and 56.6 +/- 38.4 pg/ml, respectively; controls, 17.4 +/- 9.0 pg/ml). In contrast, levels of IL-4 in serum were not increased on admission (3.4 +/- 1.2 pg/ml; controls, 2.4 +/- 0.8 pg/ml), but at day 3 a moderate and significant increase of IL-4 levels was observed (4.5 +/- 1.7 pg/ml). In conclusion, the increase of Th1 cytokine IL-12 and IFN-gamma levels during the acute phase of uncomplicated P. falciparum malaria may reflect an early and effective immune response regulated by proinflammatory Th1 cytokines, and in particular IFN-gamma may play a role in limiting progression from uncomplicated malaria to severe and life-threatening complications.     

Cytokines inhibit proliferation and insulin secretion by clonal rat insulinoma cells (RINm5F) non-synergistically and in a pertussis toxin-insensitive manner

It has been proposed that certain cytokines secreted by islet-infiltrating leukocytes may be involved in the pathogenesis of insulin-dependent diabetes mellitus. Since the cytotoxic actions by the cytokines may reflect interactions with islet cell types other than the beta-cell, in this work I have investigated the effects of different combinations of various cytokines on the proliferation and hormone content and secretion by a pure insulin-producing cell population, i.e., the clonal rat insulinoma cell line RINm5F. For this purpose RINm5F cells were exposed in culture for 1-2 days to interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and interferon alpha (IFN-alpha) at different concentrations. It was found that IL-1 beta markedly decreased the cellular content of insulin and secretion of the hormone into the culture medium, while causing a very slight inhibition of RINm5F cell proliferation. On the other hand, IFN-gamma and IFN-alpha both elicited marked decreases in proliferation and insulin content and secretion by the insulinoma cells. IL-6 and TNF-alpha were found not to affect these parameters. No additive or synergistic effects were observed when the cytokines were added in various combinations. There was no protection against the cytotoxicity of IL-1 beta, IFN-gamma or IFN-alpha by pre-treatment with pertussis toxin. From these findings it is concluded that the cytokines IL-1 beta, IFN-gamma and IFN-alpha act in a non-synergistic fashion in suppressing RINm5F cell proliferation and hormone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoimmunity is a type I interferon-deficiency syndrome corrected by ingested type I IFN via the GALT system.

Type I interferons (IFN-alpha/beta), products of the innate immune system, can modulate immune function whereas proinflammatory IFN-gamma (type II IFN), a product of the acquired immune system upregulates inflammation and enhances cell mediated immunity. We have proposed a unifying hypothesis of the origin of autoimmunity as a type I IFN immunodeficiency syndrome involving inadequate regulation of the acquired immune system product IFN-gamma by the IFN-alpha/beta innate immune system. The common theme of ingested type I IFNs in autoimmunity is inhibition of proinflammatory type II IFN systemically or at the target organ. In multiple sclerosis (MS) and insulin-dependent diabetes mellitus (IDDM) at the target organ, and in rheumatoid arthritis (RA) as a regulator of other proinflammatory cytokines, IFN-gamma is the nexus of inflammation in autoimmunity. Ingested type I IFNs counteract type II IFN, overcome the relative lack of type I IFN activity, and ameliorate autoimmunity. The administration of type I IFNs (IFN-alpha/beta) via the gut offers an exciting alternative to systemic application for overcoming the type I IFN immunodeficiency in autoimmunity. Successful use of ingested type I IFN in three separate prototypical autoimmune diseases suggests a broad antiinflammatory therapeutic profile for this technology.     

Serum cytokine changes in systemic vasculitis.

Cytokines are known to alter a number of vascular tissue cell functions. The aim of this retrospective study was to determine serum cytokine levels in patients with vasculitis and to analyse the possible relation to the severity of the disease. Tumour necrosis factor alpha (TNF alpha), interleukin-1 (IL-1)beta, IL-2, interferon (IFN)- and IFN-gamma were assayed in 33 patients with polyarteritis nodosa (PAN) or Churg and Strauss angiitis (CSA), and three with Wegener granulomatosis (WG). Serum cytokine changes were observed in most patients with active disease, i.e. before treatment was started. In the majority of patients with PAN or CSA, there was a marked increase in serum IFN-alpha and IL-2 levels, while TNF-alpha and IL-beta levels were moderately elevated. Serum IFN-gamma remained undetectable in all but one of these patients. In patients with WG, serum IFN-alpha and IL-2 levels were also elevated, whereas IL-1 beta, IFN-gamma and TNF alpha levels remained within normal limits. In paired samples of patients with PAN, IFN-alpha and IL-2 levels were significantly higher before than after treatment. These preliminary data suggest that a particular pattern of cytokine changes is associated with vasculitis and that cytokines might be involved in the pathogenesis of PAN/CSA and WG. Prospective studies are warranted to determine whether cytokines could be considered for the monitoring of disease activity and therapy.     

Type I interferon is required to mount an adaptive response to immunostimulatory DNA.

Immunostimulatory DNA sequences (ISS, CpG motifs) potently stimulate Th1 and cytotoxic T lymphocyte (CTL) responses to antigens and have thus generated considerable interest due to their potential use in immunotherapeutics. An array of cytokines are produced in response to ISS exposure, but the relative importance of each of these mediators in the stimulation of innate and adaptive ISS-induced immunity has yet to be fully investigated. To address this issue, we measured immune responses in mice with targeted deletions of the ISS-induced genes encoding IL-12 (IL-12(-/-)), IFN-gamma (IFN-gamma(-/-)), the IFN-gamma receptor (IFN-gammaR(-/-)), and the IFN-alpha/beta receptor (IFN-alpha/betaR(-/-)) after immunization with ISS-containing oligodeoxynucleotides and model antigens. IL-12(-/-) and IFN-alpha/betaR(-/-) mice were compromised in their ability to develop a cross-primed CTL response, whereas IFN-gamma(-/-) and IFN-gammaR(-/-) mice were not. In addition, lymphocytes from immunized IFN-alpha/betaR(-/-) mice had defective IFN-gamma responses to antigen restimulation. Antigen nonspecific ISS-induced B cell proliferation was normal in the four deficient strains; however, innate IL-6 production was reduced in IFN-gamma(-/-) and IFN-gammaR(-/-) splenocytes and eliminated in IFN-alpha/betaR(-/-) cells. While IL-12 production was defective in only the IFN-gamma(-/-) splenocytes, innate natural killer cell IFN-gamma synthesis was virtually absent in the IL-12(-/-) and IFN-alpha/betaR(-/-) mice. Thus, while IFN-alpha/beta, IFN-gamma, and IL-12 each play important and distinct roles in the development of the innate and adaptive immune responses to ISS, IFN-alpha/beta is a particularly crucial and currently under-appreciated factor in this system.     

Immunomodulation and therapeutic effects of the oral use of interferon-alpha: mechanism of action.

It is now well accepted that type 1 interferons (IFNs), IFN-alpha and IFN-beta, in addition to being molecules with powerful antiviral activity, play a critical role in modulating immune responses to foreign and self-antigens. This review of the literature documents the immunomodulatory effects of IFN-alpha and discusses its position and importance in the cytokine cascade. In addition, this review attempts to organize the literature describing local and systemic immunomodulatory effects of orally administered low doses of IFN-alpha, and provide a physiological explanation for the mechanism of action. Evidence suggests that, early in the process of antigen presentation to T helper (Th) cells, IFN-alpha derived principally from the antigen-presenting cells (APC) provides an important signal for Th precursor differentiation in favor of a Th1 immune response. IFN-alpha, perhaps via upregulation of the high-alphaffinity interleukin-12beta1/beta2 (IL-12beta1/beta2) receptor, renders Th1 cells responsive to IL-12 resulting in production of high levels of IFN-gamma crucial to the development of Th1 immune responses. In addition to being instrumental in the development of Th1 immune responses, IFN-alpha appears to be the major cytokine responsible for the amplification of the CD8+ T cell response and resistance to viral infections. Orally administered IFN-alpha induces similar Th1 cytokine responses in buccal mucosal lymph nodes (LN), including upregulation of IFN-gamma expression and downregulation of IL-4. Moreover, reports of systemic immune effects such as decreased autoimmune responses, increased antiviral and antibacterial responses, and generalized immune function changes after oral IFN-alpha administration are consistent with the known immunomodulatory role of IFN-alpha in a physiological setting. Responses to orally administered low doses of IFN-alpha also adhere to the principle of low-dose priming and high-dose anergy that dictates the cellular and cytokine responses to exogenously added cytokines both in vivo and in vitro. These observations collectively suggest that IFN-alpha administered to mucosal-associated immune tissue replicates the known physiological role of IFN-alpha, including regulation of CD4+ Th1 immunomodulatory cells and activation of CD8+ effector cells, which are both crucial to development of protective immune responses. What remains to be determined is how local mucosal immune responses to IFN-alpha given orally are translated into systemic immune responses and resistance to disease. This important question, the answer to which will have profound implications for new immunotherapies for immune-based diseases, is the focus of current research.     

IL-16 inhibits IL-5 production by antigen-stimulated T cells in atopic subjects

BACKGROUND: We have previously shown increased expression of the CD4+ cell chemoattractant IL-16 at sites of airway allergic inflammation. Little is known about the significance of IL-16 in allergic inflammation and its role in allergen-driven T-cell cytokine responses. Because IL-16 interacts specifically with CD4+ T cells, we hypothesized that IL-16 released at sites of inflammation may modulate the pattern of cytokines produced by CD4+ T cells. OBJECTIVE: We investigated the effects of exogenous rhIL-16 on cytokine production of PBMCs from atopic and nonatopic subjects in response to antigen and PHA. METHODS: Primary cultures of freshly isolated PBMCs from ragweed-sensitive atopic subjects and nonatopic subjects were stimulated with ragweed or PHA in the presence or absence of rhIL-16. Supernatant levels of IL-4, IL-5, and IFN-gamma were determined by means of ELISA at different time points between 2 and 6 days. Effects of IL-16 on antigen-induced cellular proliferative responses were determined. RESULTS: No IL-4 protein was detected after antigen stimulation of PBMCs from atopic subjects, whereas significant levels of IL-5 were measured on day 6 (median, 534.9 pg/mL). IL-5 secretion was abolished in PBMC cultures depleted of CD4+ cells. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly reduced the amount of IL-5 released (median, 99.8 pg/mL; P <.001). Detectable levels of IFN-gamma (median, 53.3 pg/mL) were identified after antigen stimulation. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly increased IFN-gamma levels (median, 255.6 pg/mL; P <.05). Effects of rhIL-16 appear to be specific for antigen-stimulated PBMCs in atopic subjects because rhIL-16 did not alter IL-5 or IFN-gamma production in response to PHA nor did rhIL-16 alter cytokine production in nonatopic normal subjects. CONCLUSION: These studies suggest that IL-16 can play a role in regulating the production of cytokines seen in allergic states in response to antigen.     

Autoimmunity to a pathogenic retinal antigen begins as a balanced cytokine response that polarizes towards type 1 in a disease-susceptible and towards type 2 in a disease-resistant genotype.

Susceptible, but not resistant, strains of rodents immunized for induction of experimental autoimmune uveitis (EAU) with the uveitogenic protein interphotoreceptor retinoid-binding protein (IRBP) exhibit a type 1 response at the time of disease expression. Here we investigate the evolution of this response using the prototypic EAU-susceptible and EAU-resistant mouse strains, B10.A and BALB/c. Disease severity and IRBP-specific responses (proliferation, cytokines and antibody isotypes) were evaluated 7, 14 and 21 days after uveitogenic immunization. B10.A mice initially exhibited an IgG1-dominated antibody response, and their lymph node cells produced IL-4 and IL-5 in addition to IFN-gamma. On day 14 and 21, however, the IgG2a isotype became predominant, and the primed lymph node cells produced mainly IFN-gamma and IL-12. B10.A mice developed EAU before day 14. BALB/c mice initially produced IL-12 and IFN-gamma in addition to IL-5, IL-4 and IL-10. At later time points IL-12 and IFN-gamma production diminished, and IL-4, IL-5 and IL-10 increased. An IgG1-dominated antibody response was maintained throughout. BALB/c mice failed to develop EAU even at day 21. Thus, both susceptible and resistant genotypes initially mount a balanced, type 0-like cytokine response to a uveitogenic challenge, that subsequently polarizes towards type 1 in the susceptible strain and towards type 2 in the resistant strain.     

Interferon-gamma inhibits in vitro mobilization of eosinophils by interleukin-5

BACKGROUND: Th2 cytokines play pivotal roles in allergic inflammation, including eosinophilia, and their actions are antagonized by Th1 cytokines, conferring them therapeutic potential. METHODS: In this study, we examined the ability of a number of cytokines to suppress the activation of eosinophils that function as effector cells for allergic airway diseases. RESULTS: Interleukin (IL)-5, IL-6, and tumor necrosis factor (TNF) induced an eosinophil shape change, whereas interferon (IFN)-gamma significantly inhibited the shape change. Other cytokines, including IL-1beta, IL-4, IL-10 and IL-13, had little or only slightly enhancing or reducing effects on the shape change. We further analyzed the IFN-gamma effect, showing that pretreatment with IFN-gamma strongly suppressed IL-5-induced eosinophil shape change, and cycloheximide (CHX) abrogated the suppression by IFN-gamma, suggesting that new protein synthesis is required for the inhibitory effect by this cytokine. In agreement with these results, IFN-gamma blocked the eosinophil migration and ERK phophorylation induced by IL-5, and the addition of CHX restored eosinophil chemotaxis. CONCLUSIONS: Collectively, IFN-gamma may attenuate eosinophilic inflammation by directly negating eosinophil mobilization.