Immunomodulatory activities of IFN-gamma1b in combination with type I IFN: implications for the use of IFN-gamma1b in the treatment of chronic HCV infections.

The standard of care for chronic hepatitis C, pegylated interferon-alpha (IFN-alpha) and ribavirin (RBV), causes a sustained virologic response (SVR) in approximately 50% of patients. SVR is correlated with innate and adaptive immune system responses, such as natural killer (NK) cell activation, production of IFN-alpha from immature plasmocytoid dendritic cells (pDC), and polarization of CD4(+) cells to a T helper 1 (Th1) cell phenotype. To examine how these immunologic responses vary with currently available regimens for chronic hepatitis C, cell populations purified from human peripheral blood mononuclear cells (PBMC) were treated with the clinically available combinations of pegylated IFN-alpha2b (PEG-IFN-alpha2b) + RBV, IFN-alphacon1 + RBV, or IFN- alphacon1 + IFN-gamma1b, and activation of cellular immune system components was monitored. The magnitude of NK cell activation depended on regimen, with IFN-alphacon1 + IFN-gamma1b > IFN-alphacon1 + RBV > PEG-IFN- alphaa2b + RBV. The maximum human serum concentrations of IFN-alphacon1 + IFN-gamma1b saturated NK cell activation, whereas the maximum human serum concentrations of IFN-alphacon1 + RBV or PEG-IFN-alpha2b + RBV did not. IFN-gamma1b also enhanced the production of IFN-alpha from immature pDCs, which are the dominant source of IFN-alpha upon viral infection. The rank order for induction of Th1 cell phenotype and repression of Th2 cell phenotype by the cocktails described was identical to that observed for NK cell activation. Additionally, IFN- gamma1b suppressed the ability of the hepatitis C virus (HCV) NS4 protein to enhance monocyte secretion of interleukin- 10 (IL-10), a cytokine whose expression level is correlated with viral persistence. These results suggest that addition of IFN-gamma1b to HCV treatment regimens may provide unique benefits.     

Interleukin-12 p40/p70 ratio and in vivo responsiveness to IFN-alpha treatment in chronic hepatitis C.

To evaluate the relationship between cytokine balance and responsiveness to interferon-alpha (IFN-alpha), we investigated the production of IFN-gamma, interleukin-10 (IL-10), IL-12 p70, and IL-12 p40 by peripheral blood mononuclear cell (PBMC) cultures from patients with chronic hepatitis C (CHC) before and after 1 year of IFN-alpha treatment. Before the therapy, responder (R) patients exhibited lower IFN-gamma release, higher IL-10 production, and higher values of the IL12 p40/p70 ratio compared with nonresponders (NR). Increased sensitivity to the effects of IL-12 and IL-10, as well as higher IL-12-dependent IFN-gamma secretion, were also found in the R subset. After IFN-alpha therapy, an increase in IFN-gamma production and a decrease in the IL-12 p40/p70 ratio were observed in R patients, whereas opposite results were obtained in the NR group. Finally, the therapy induced downregulation of IL-10 production and cell responsiveness to recombinant IL-12 in all patients. These findings imply that predominance of a T helper 2 (Th2) cytokine profile in CHC patients favors the beneficial effects of IFN-alpha, thus suggesting a therapeutic role for Th1-driven stimulation of immune response. The findings also stress the primary importance of the IL-12 p40 and p70 balance in the modulation of immune responses to hepatitis C virus (HCV).     

Ribavirin increases mitogen- and antigen-induced expression of CD40L on CD4+ T cells in vivo

Here, CD40L expression and cytokine production have been analysed in peripheral blood cells from orthotopic liver transplantation (OLT) recipients treated with ribavirin for recurrent chronic hepatitis C. The study included 18 OLT recipients treated with ribavirin, eight control OLT recipients and 10 healthy controls. FACS analysis showed that baseline expression of CD40L was not different between ribavirin-treated patients and controls. In contrast, after stimulation with both HCV core antigen and phorbol myristate acetate (PMA) plus ionomycin (IO), the expression of CD40L on CD4 lymphocytes was significantly higher in the ribavirin group compared with controls. In the ribavirin group, the increased expression of CD40L significantly correlated with reduction of HCV RNA levels with respect to pretreatment values. Finally, ribavirin treatment was not associated with modification of PMA-IO-induced cytokine production by T lymphocytes and interleukin (IL)-1beta and tumour necrosis-alpha (TNF)-alpha production by CD40L-stimulated monocytes. In conclusion, these data indicate that ribavirin -upmodulates CD40L expression on CD4 T cells, a property which may account in part for its ability to enhance the antiviral activity of interferon-alpha in the treatment of chronic HCV infection.     

IFN-alpha2b increases interleukin-10 expression in primary activated human CD8+ T cells.

Interleukin-10 (IL-10) is a multifunctional cytokine with diverse effects on most hematopoietic cell types. It appears the principal function of IL-10 is to limit and ultimately terminate inflammatory response. We demonstrate here that interferon-alpha2b (IFN-alpha) increases the expression of IL-10 in activated primary CD8(+) T cells. Optimal induction of mRNA expression and protein synthesis was observed when IFN-alpha was added to cells activated by the combination of anti-CD3 monoclonal antibody (mAb) and IL-2. Maximal stimulation of IL-10 protein production was observed after prolonged incubation periods (48-72 h). No effects were observed on the production of IL-4, whereas IFN-gamma was produced with a faster kinetics than an untreated control. Our data indicate that IFN-alpha promotes the development of a CD8(+) T cell population with enhanced anti-inflammatory activity, which may play a critical role in the regulation of a proper immune response.     

In vitro responsiveness to IL-18 in combination with IL-12 or IL-2 by PBMC from patients with bronchial asthma and atopic dermatitis

Interleukin (IL)-18 is a proinflammatory cytokine and is now recognized as an important regulator of both helper T cells (Th) 1 and 2 cytokine production. An increased IL-18 secretion has been reported in patients with allergic disorders. It is predominantly produced by activated macrophages, and synergizes with IL-12 and IL-2 to induce IFN-gamma synthesis, thereby promoting Th1 cytokine response. Paradoxically, IL-18, by itself, strongly induces immunoglobulin (Ig) E and allergic inflammation, indicating a role for IL-18 in promoting Th2 response. We investigated the inducing effect in vitro of combining IL-18 and Il-12 or Il-2 on Th1- and Th2-type cytokines production by peripheral blood mononuclear cells (PBMC) from patients with allergic diseases. PBMC derived from 44 allergic patients [23 bronchial asthma (BA) and 21 atopic dermatitis (AD)] and 20 healthy controls were cultured with IL-18 in the presence of phytohemagglutinin (PHA) and IL-12 or IL-2. The levels of IFN-gamma, IL-13, and IL-4 in the culture supernatants were measured using enzymatic immunoassaying. IFN-gamma production was detected in all cultures from nonallergic controls stimulated with IL-18 in the presence of IL-12; however, the results for five BA patients and five AD patients were under the detection limit for IFN-gamma. In collaboration with IL-2, IL-18 was able to induce IFN-gamma production by PBMCs from all nonallergic controls and all allergic patients, with the exception of one AD patient. Synergistic induction of IL-13 production was found in cultures with IL-18 + IL-2, and the IL-13 induction was significantly increased in BA patients when compared with that in nonallergic controls (P = 0.006). The stimulation by IL-18, even in combination with IL-2, failed to induce IL-4 production by PBMC from both nonallergic controls and allergic patients. Although the induction of IFN-gamma by IL-18 + IL-12 was impaired in around a quarter of the allergic patients, the impairment of the IFN-gamma production was completely restored by IL-2 in the presence of IL-18. Thus, IL-18 enhances IFN-gamma production through an IL-12-dependent pathway and exhibits synergism when combined with IL-2 in terms of enhanced IL-13 and IFN-gamma production, suggesting the involvement of IL-18/IL-12/IL-2 pathway in modulating Th1/Th2 cytokine response.     

Modulation of the IL-12/IFN-gamma axis by IFN-alpha therapy for hepatitis C

Although IFN-alpha forms the foundation of therapy for chronic hepatitis C, only a minority of patients has a sustained response to IFN-alpha alone. The antiviral activities of IFN-alpha formed the rationale for its use in viral hepatitis. However, IFN-alpha and the other Type I IFNs are also pleiotropic immune regulators. Type I IFNs can promote IFN-gamma production by activating STAT4 but can also inhibit production of IL-12, a potent activator of STAT4 and IFN-gamma production. The efficacy of IFN-alpha in the treatment of hepatitis C may therefore depend in part on the balance of IFN-gamma-inducing and IL-12-suppressing effects. We characterized the effects of pegylated IFN-alpha therapy for hepatitis C on the capacity of patients' PBMC to produce IL-12 and IFN-gamma ex vivo. Cells from patients with a sustained virological response to therapy had significantly greater levels of IFN-alpha-driven IFN-gamma production prior to treatment than those from nonresponding patients. No differences in pretreatment IL-12 productive capacity were seen between patient groups. However, therapy with IFN-alpha led to suppression of inducible IL-12 production throughout the course of therapy in both groups of patients.     

IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice

Interferon-gamma (IFN-gamma) is an important immunomodulatory and pleiotropic cytokine produced, primarily, by activated T lymphocytes and natural killer (NK) cells. We have devised a nitric oxide (NO)-based bioassay for mouse IFN-gamma using resident peritoneal exudate cells (PECs) from C57BL/6 mice. Comparison with three existing bioassays demonstrated that this assay was very sensitive and detected IFN-gamma in the linear range of approximately 0.03-0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-alpha/beta and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination with IFN-gamma, did not greatly modulate NO levels produced by resident peritoneal exudate cells. The presence of exogenous NO(3)(-) and H(2)O(2) did not interfere with the IFN-gamma induced NO production and detection. We also showed that the effect of lipopolysaccharide (LPS), which may be present in samples, could be suppressed by the use of Polymyxin B in the bioassay. The high sensitivity of the bioassay permitted the detection of low amounts of IFN-gamma in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-gamma amounts in supernatants of activated T cells. The increase in IFN-gamma production by activated T cells in response to CD28 costimulation was approximately 3-fold by this bioassay and approximately 5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-gamma.     

Lack of evidence for the Th2 predominance in patients with chronic hepatitis C

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-gamma/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-gamma + or IL-2 +) was significantly increased in CD4 +, CD8 +, 'naive'-CD45RA + and 'memory'-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-gamma-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.     

Identification of the association of CD28+CD244+ Tc17/IFN-γ cells with chronic hepatitis C virus infection

CD8⁺ T cells play multiple and complex immunological roles including antiviral, regulatory, and exhaustive effects in hepatitis C virus (HCV) infected patients. Some CD8⁺ T-cell subsets were confirmed to be closely related to HCV infection such as T_CM, T_EM, T_EMRA, Tc17, and CD8⁺ Treg. Herein, we report a new subset of interleukin (IL)-17/interferon (IFN)-γ producing CD8⁺ T (Tc17/IFN-γ) cells that markedly correlate with CD28⁺CD244⁺ cells, IL-17 levels, and HCV RNA in HCV patients. During early treatment with peg-IFN-a2a plus ribavirin, the imbalance of these Tc17/IFN-γ cells could be partially restored, together with normalized serum alanine aminotransferase but not aspartate transaminase. Also, we analyzed the dynamic change of the percentage of this T cells subset in patients with different outcome after 4-week course of treatment with peg-IFN-a2a plus ribavirin and found that the percentage of CD8⁺CD28⁺CD244⁺ T cells significantly decreased in recovered patients but not in nonrecovered patients. In vitro, CD28⁺CD244⁺ T cells were the only CD8⁺ T-cell group that secreted both IL-17 and IFN-γ in this axis and blockade with anti-CD244 antibodies significantly reduced cytokine production. Taken together, this study demonstrates that the frequency and regulatory functions of CD28⁺CD244⁺ Tc17/IFN-γ cells may play an important role in persistent HCV infection.     

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-gamma production by allergen and PPD specific T cells

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-gamma. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-gamma production. Synthesis of IFN-gamma can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-gamma and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-gamma were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-gamma levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.     

IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells.

Interleukin-27 (IL-27) supports proliferation of naive CD4(+) T cells and enhances interferon-gamma (IFN-gamma) production by activated T cells and natural killer (NK) cells. We report here that IL-27 induces Stat1 and Stat3 phosphorylation and activation in human and murine cell lines and primary human T cells. IL-27 also induces T-Bet, a Stat1-dependent gene crucial to Th1 cell commitment. Similarly, IFN-alpha activates Stat1 and Stat3 and T-Bet expression in naive T cells. Induction of T-Bet results in upregulation of IL-12Rbeta2 on naive T cells, which is essential for responsiveness to IL-12 and differentiation to a Th1 phenotype. Both IL-27 and IFN-alpha induce expression of IL-12Rbeta2 in T cells. In contrast, IFN-gamma, which activates Stat1 but not Stat3, induces expression of T-Bet but not IL-12Rbeta2 in naive T cells. We propose that IL-27 and IFN-alpha are important for early Th1 commitment and act upstream of IL-12 and IFN-gamma in this pathway.     

Enhancing the response to interferon-alpha.

BACKGROUND: Though initially recognized as antiviral agents, it was soon demonstrated that certain neoplasms were particularly sensitive to interferon-alpha (IFN-alpha). Indeed, the initial success of systemic IFN-alpha treatment in AIDS-associated Kaposi's sarcoma (AIDS-KS) occurred before identification of the human immunodeficiency virus (HIV) and in the absence of any coherent view of KS pathogenesis. With a more comprehensive understanding how KS develops and which circumstances provide an increased virulence of this neoplasm in HIV-infected persons, a more subtle rationale for IFN-alpha treatment arose regarding the disorder of the endogenous IFN-system in HIV-positive individuals. Until recently IFN-alpha was the only therapy available for patients with chronic hepatitis C (CHC). However, no more than 30% of these patients show a sustained virological response. Initial therapy with a combination therapy of IFN-alpha and ribavirin turned out to be more effective than treatment with IFN-alpha alone. To ameliorate response rates in antiviral IFN-therapy a profound understanding of viral dynamics, as well as immunological conditions associated with viral persistence, seems to be essential. Within a conference of the European Society of Clinical Virology (ESCV), which took place in Hamburg from August 30 to September 2, 1998, and was entitled 'Progress in Clinical Virology IV', a satellite symposium was organized to evaluate the clinical results of special antiviral treatment options with IFN-alpha, to analyze treatment failures with this cytokine and to ameliorate future strategies of IFN-alpha therapy. It focussed on HIV-related complications as coinfection with hepatitis C virus (HCV) and AIDS-KS, respectively. METHODS: A kinetic model of HCV infection based on principles established in studying HIV-1 infection was presented which is predictive for the outcome of IFN-alpha treatment. It involves different rates of velocity and compares the rates of acute clearance after different dosages of IFN-alpha application. Using the hypothesis to fit the changes in serum HCV RNA measured in a set of patients, it was found that 5 mIU daily dosing on average blocks 81% of HCV production/release, whereas 10 or 15 mIU blocks about 95% of HCV production/release. RESULTS: Only recently clinical data revealed a greater benefit of combination therapy with IFN-alpha and ribavirin compared to IFN-alpha alone in patients with chronic hepatitis C. In 345 CHC patients relapsing after pretreatment with IFN-alpha monotherapy, sustained response was achieved in a 10-fold higher degree with a combination of IFN and ribavirin compared to patients retreated with IFN alone. In 1775 treatment-naive patients with CHC, response rates to the combination therapy was significantly higher in all patient groups with more than 60% of sustained virological response in patients with genotype 2 and 3, while patients with genotype 1 (poorer prognosis) benefit from extended combination treatment duration from 24 to 48 weeks (17 versus 29% of sustained virological response), respectively. CONCLUSIONS: As viral dynamics on one side and host immune response on the other feature as two landmarks on which the manifestation of viral persistence and chronic viral infections is established, some similarities of HCV and HIV disease are striking. An unusual endogenous IFN-alpha system is associated with both infections and is a negative prognostic factor to response to treatment with IFN-alpha in CHC as well as AIDS-KS. The consequences for treatment options with IFN are a combination with ribavirin in CHC and a graduated systemic treatment schedule in AIDS-KS starting with IFN-treatment in early disease followed by chemotherapy in advanced stages of KS.     

Intrahepatic MxA and PKR protein expression in chronic hepatitis C virus infection

The therapeutic effect of interferon-alpha and ribavirin in the treatment of chronic hepatitis C viral infection is limited. To identify patient characteristics that may predict responsiveness to treatment, the intrahepatic protein expression of two directly induced IFN-alpha effector proteins, MxA and PKR, were studied. Forty liver biopsy samples from patients with a variety of chronic liver diseases were stained for MxA and PKR protein using immunohistochemical techniques. In a HCV patient cohort, 30 liver biopsies were stained for MxA and PKR protein prior to treatment with IFN-alpha and ribavirin. PKR protein expression was not upregulated in viral liver disease. In contrast, MxA protein expression was significantly upregulated in viral liver disease (P = 0.005). In chronic HCV liver disease, moderate to strong cytoplasmic expression of MxA protein was observed in hepatocytes and monocytes, indicating endogenous hepatocellular IFN-alpha pathway activation. In the HCV patient cohort treated with combination therapy, strong pre-treatment MxA hepatocyte expression was predictive of a non-response to treatment (odds ratio 9.33; P = 0.01; 95% confidence interval 1.63-53.2). This effect was independent of HCV genotype and viral load. It is concluded that pretreatment hepatocellular MxA expression may become a useful predictor of response to combination treatment with IFN-alpha and ribavirin.     

Interleukin (IL)-4, IL-10, IL-18 and IFN-gamma cytokines pattern in patients with combined hepatitis C virus and Schistosoma mansoni infections

Schistosoma mansoni infection is characterized by a strong T-helper type 2 (Th2) cell-associated immune response, but in the case of viral infection, it is associated with interferon-gamma (IFN-gamma) increase and induction of Th1 immune response. Few data are available about the immune response of cases infected with combined hepatitis C virus (HCV) and schistosomiasis. Thus, the investigation of the cytokine pattern in patients coinfected with both HCV and Schistosoma mansoni was our rationale. This study included four patient groups: Group 1 included 20 patients infected with chronic HCV, Group 2 included 15 patients infected with schistosomiasis alone, Group 3 included 20 patients with chronic HCV and schistosomiasis and Group 4 included 15 healthy control individuals with matched age and sex. Serum levels of IFN-gamma, interleukin (IL)-4, IL-10 and IL-18 were measured in all groups by enzyme-linked immunosorbent assay. The results showed that the patients infected with HCV had significantly higher serum levels of IFN-gamma and IL-18 compared with the controls and with the patients with schistosomiasis and coinfection (P < 0.001). On the other hand, serum levels of IL-4 and IL-10 were significantly higher in patients with schistosomiasis and coinfection compared with the control group (P < 0.001 and 0.0001, respectively) and with the HCV patients (P < 0.05 and P < 0.001, respectively). A significant increase in serum levels of IL-4 and IL-10 was also found in HCV patients compared with the control (P < 0.05). Schistosomiasis appears to induce a Th2 cytokine profile, with increase in serum levels of IL-4 and IL-10, even in the presence of HCV coinfection. In conclusion, schistosomiasis may downregulate the stimulatory effect of HCV on Th1 cytokines and this may lead to the chronicity of HCV infection in coinfected patients.