Alan D. Levine

IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

IL-6 Signaling in Psoriasis Prevents Immune Suppression by Regulatory T Cells

T memory/effector cells (Tmem/eff) isolated from psoriatic patients are chronically activated and poorly suppressed by regulatory T cells (Treg). The proinflammatory cytokine IL-6, which signals through Stat3, allows escape of Tmem/eff cells from Treg-mediated suppression in a murine system. We show here that IL-6 protein is markedly elevated and most highly expressed by CD31+ endothelial cells and CD11c+ dermal dendritic cells (DCs) in lesional psoriatic skin. We hypothesized that exposure to high IL-6 in lesional tissue may lead to the dampened Treg function observed in psoriasis patients. Indeed, we found that IL-6, but not other Stat3-activating cytokines, was necessary and sufficient to reverse human T cell suppression by Treg in an in vitro model using activated DCs as a source of IL-6. IL-6Rα and gp130 expression was significantly elevated in psoriatic effector T cells compared with normal controls. Overall, IL-6Rα expression on Treg exceeded that of effector T cells, and both populations phosphorylated Stat3 in response to IL-6. Phosphorylation of Stat3 in T cells contributes to Th17 differentiation and we identify cells within lesional tissue that coexpress CD3, IL-17, and IL-6, indicating that Th17 cells are present in vivo within the psoriatic Tmem/eff population and contribute to IL-6-mediated resistance to Treg suppression. Taken together, T lymphocytes trafficking into lesional psoriatic skin encounter high IL-6 from endothelial cells, DCs, and Th17 cells, enabling cutaneous T cell escape from Treg suppression and Th17 participation in inflammation. Targeting IL-6 signaling pathways in psoriasis may rebalance Treg/T effector activity and ameliorate disease.

Interleukin-10 Controls the Onset of Irreversible Septic Shock

ABSTRACT Lethality from sepsis is believed to be mediated by a proinflammatory cytokine cascade, yet blocking the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) fails to prevent mortality in human disease and a mouse model of sepsis induced by cecal ligation and puncture (CLP). The role of the antiinflammatory cytokine IL-10 in the CLP model of sepsis is unclear, with either protective or harmful effects demonstrated, depending upon the time of intervention. We therefore hypothesize that IL-10 functions as a temporal regulator of the transition from early reversible sepsis to the late phase of irreversible shock. Transition from reversible sepsis to irreversible shock in the CLP model was defined as the time when removal of the necrotic cecum by rescue surgery is no longer effective. We subjected IL-10-deficient (IL-10−/−) and wild-type (IL-10+/+) mice to CLP and monitored the progression of sepsis, the onset of irreversible shock, and mortality. Onset of lethality in IL-10−/− mice occurred significantly earlier than in IL-10+/+ mice and was associated with 15-fold-higher serum levels of TNF-α and IL-6. Consistent with these findings, the efficacy of rescue surgery after lethal CLP is lost 10 h earlier in IL-10−/− mice than in IL-10+/+ mice. Treatment with recombinant human IL-10 5 h after CLP significantly improved survival and lengthened the therapeutic window for rescue surgery in both strains of mice. These results demonstrate that IL-10 controls the onset of irreversible septic shock after CLP.

GERMINAL CENTER T CELLS ARE DISTINCT HELPER-INDUCER T CELLS

Germinal centers (GCs) contain a significant number of CD4+ T cells, but what role these T cells may play in the development of GC B cells has not been determined. To gain insight into their role, we studied the phenotype of GC T cells and the lymphokines secreted by GC T cells isolated from human tonsils obtained after tonsillectomies. In addition to confirming that a large fraction of GC T cells are Leu-7(CD57)+ and Leu-8-, we found that they have no binding sites for peanut agglutinin. Furthermore, we found that they are CD45RA- and CD45R0+, the phenotype of helper-inducer T cells. We also found that Leu-7(CD57)+ cells display CD69, a phenotypic marker of very early cell activation, but do not display three other markers of cell activation: CD25 [interleukin-2 (IL-2) receptor], CD71 (transferrin receptor), and DR. When isolated, Leu-7(CD57)+ cells were stimulated in vitro with a mitogen that can induce peripheral blood T cells with the helper-inducer phenotype to produce various cytokines, Leu-7(CD57)+ cells did not produce IL-2, interleukin-4 (IL-4), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) in significant amounts. Taken together, GC T cells from a distinct subpopulation of T cells with helper-inducer phenotype by their histologic location, by their surface phenotype, and by their ability to produce lymphokines. This finding is consistent with the possibility that GC T cells have been selectively recruited to actively help B cells develop in GCs.

Circulating CD4+ and CD8+ T cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation

Inflammatory bowel disease (IBD) is characterized by damage to the gut mucosa and systemic inflammation. We sought to evaluate the role of chronic inflammation on circulating T‐cell activation in human subjects with Crohns disease and ulcerative colitis. We studied 54 patients with IBD and 28 healthy controls. T‐cell activation and cycling were assessed in whole blood samples by flow cytometry. Levels of lipopolysaccharide (LPS) were measured in serum by Limulus amoebocyte lysate assay, and plasma levels of inflammatory markers and LPS‐binding proteins were measured by ELISA. The proportions of circulating CD4+ and CD8+ T lymphocytes in cycle (Ki67+) are increased in patients with IBD compared with these proportions in controls. CD8+ T cells from patients with IBD are also enriched for cells that expressed CD38 and HLA‐DR, and proportions of these cells are related to plasma levels of interleukin‐6 and C‐reactive protein in these patients. Intracellular interleukin‐2 and interferon‐γ levels were elevated in resting and polyclonally activated CD4+ and CD8+ T cells in patients with IBD when compared with levels from healthy controls. Surprisingly, we did not find increased levels of LPS in the serum of patients with IBD. We did, however, find a signature of recent microbial translocation, as levels of LPS‐binding protein are increased in the plasma of patients with IBD compared with plasma levels in healthy controls; LPS‐binding protein levels are also directly related to proportions of CD38 HLA‐DR‐expressing CD4+ and CD8+ T cells. Local damage to the gastrointestinal tract in IBD may result in systemic inflammation and T‐cell activation.

Mechanisms of natural tolerance in the intestine: Implications for inflammatory bowel disease

Tolerance, the regulated inability to respond to a specific immunologic stimulant, is a physiological event important to normal immune function. Just as loss of tolerance to self-proteins results in autoimmune diseases, we assert that loss of tolerance to commensal flora in the intestinal lumen leads to inflammatory bowel disease (IBD). Mechanisms through which the mucosal immune system establishes and remains hyporesponsive toward the presence of food proteins and commensal flora, which we define as natural tolerance, are discussed. In addition to the contributions by commensal flora, the innate host defense and the adaptive immune systems promote natural tolerance to sustain normal mucosal homeostasis. Understanding the molecular and cellular events that mediate natural tolerance will lead to more advanced insights into IBD pathogenesis and improved therapeutic options.

REDOX regulation of IL-13 signaling in intestinal epithelial cells: usage of alternate pathways mediates distinct gene expression patterns.

In the classic view interleukin-13 (IL-13) binds to a heterodimer protein complex of the IL-13Ralpha1 and IL-4Ralpha chains and signals through a Janus kinase 1 (JAK1)-signal transducer and activator of transcription 6 (STAT6) mechanism. We recently reported that IL-13 also signals through the IL-13Ralpha2 chain initiating all three mitogen activated protein kinase (MAPK) pathways, and the relative expression of IL-13Ralpha1 and IL-13Ralpha2 modulates one anothers transduction pathway. Therefore we investigated whether generation of reactive oxygen species (ROS) as second messengers may serve as a common nexus between these two pathways emanating from the individual IL-13 receptor chains in intestinal epithelial cells (IEC). IL-13 stimulates intracellular ROS synthesis within 5min via IL-13Ralpha1-JAK1-STAT6- and IL-13Ralpha2-MEK1/2-ERK1/2-dependent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1). IL-13-induced ROS generation in turn positively regulates phosphorylation of ERK1/2 and STAT6, yielding a feed forward amplification loop. IL-13 also stimulates the stable, long-term gene expression of two other NADPH oxidases, NOX-4 and DUOX-2, which along with constitutive NOX-1, might facilitate elevated, continuous production of ROS in IL-13-activated IEC. The contribution of each signal transduction pathway initiated by IL-13 engagement to such biological functions as wound healing, inflammation, and apoptosis was mapped for representative, responsive genes. Distinct usage patterns were observed, demonstrating not only that IL-13 signal transduction through STAT6, MAPK, and ROS is regulated in both an antagonistic and cyclic fashion, but also that each pathway plays a specific role in modulating the wound healing and anti-apoptotic capabilities of the intestinal epithelium.

Mononuclear cells from patients with the hyper-IgE syndrome produce little IgE when they are stimulated with recombinant human interleukin-4☆

To investigate whether B cells from patients with the hyper-IgE syndrome are more sensitive to the effects of interleukin-4 in vitro than B cells of normal or atopic individuals, we stimulated blood mononuclear cells (MNC) with varying doses of recombinant human interleukin 4 (rhIL-4) and measured supernatant IgE concentrations after 18 days of culture. Geometric mean spontaneous IgE synthesis after 18 days of culture without rhIL-4 was low (less than 3 ng/ml) and similar for MNCs from nine patients with the hyper-IgE syndrome, nine atopic and nine normal subjects. As found in our previous studies, MNCs from the nine atopic and the nine normal donors produced significant and similar quantities of IgE (geometric mean maximum IgE, 25.2 and 18.7 ng/ml, respectively) when MNCs were stimulated with rhIL-4. MNCs from both donor groups had similar sensitivity to the concentration of IL-4 eliciting the IgE response. In striking contrast, MNCs from the nine patients with the hyper-IgE syndrome failed to produce significant IgE over that produced spontaneously when MNCs were stimulated by a wide range of rhIL-4 concentrations. Coculture of B cell-enriched subpopulations from patients with the hyper-IgE syndrome with T cell-enriched subpopulations from nonatopic and atopic donors failed to restore responsiveness to rhIL-4. The addition of anti-CD40 monoclonal antibody to MNC cultures did result in enhancement of rhIL-4 IgE synthesis by MNCs from patients with the hyper-IgE syndrome, but the concentration of anti-CD40 required to elicit this enhancement was tenfold higher than for control MNCs.(ABSTRACT TRUNCATED AT 250 WORDS)

A cell culture system that enhances mononuclear cell IgE synthesis induced by recombinant human interleukin-4

A new culture system is described in which recombinant human interleukin-4 (rhIL-4) consistently induces the synthesis of large quantities of IgE by human blood mononuclear cells (MNC). Unfractionated MNC were cultured in complete Iscoves modified Dulbeccos medium (C-IMDM), composed of IMDM enriched with human transferrin, bovine insulin, bovine serum albumin, oleic acid, palmitic acid, linoleic acid, and fetal calf serum (FCS). Under these culture conditions, MNC from four donors synthesized mean quantities of IgE of 76 ng/ml at plateau after stimulation with rhIL-4 in concentrations ranging from 0.04 to 80 ng/ml (plateau rhIL-4 concentrations were 5 ng/ml or greater). In contrast, rhIL-4 failed to induce significant IgE synthesis at any of those doses of rhIL-4 in parallel MNC cultures performed in RPMI 1640 supplemented with FCS (RPMI 1640). Additional optimal conditions for the induction of IgE synthesis in this system were a MNC concentration of 1-2 X 10(6)/ml and a culture time of 18 days. Variability was noted in the amount of IgE produced by different donors (CV 0.22) and by the same donor when tested on different occasions (mean CV 0.21), but no donors MNC failed to produce significant IgE in response to rhIL-4 when cultured in C-IMDM. The geometric mean IgE production induced by optimal IL-4 concentrations for the entire group of 16 subjects was 36.8 ng/ml IgE, with the lowest day 18 mean IgE concentration for any donor being 10.6 ng/ml and the highest 372.2 ng/ml. The enhanced rhIL-4-induced IgE synthesis supported by C-IMDM was due to the combined effects of the added enrichment factors and not to differences in the viabilities of MNC cultured in C-IMDM and RPMI 1640. This culture system will alleviate the problems of inconsistent and low quantities of IgE induced by IL-4 that confound most current culture systems used to examine rhIL-4-induced IgE synthesis. It will, thereby, facilitate further investigation of the regulation of human IgE synthesis.

Interleukin 10 extends the effectiveness of standard therapy during late sepsis with serum interleukin 6 levels predicting outcome.

Patients with septic shock often display features of T cell hyporesponsiveness and immune suppression, which, if persistent, are associated with increased mortality. In the murine cecal ligation and puncture (CLP) model of sepsis, we previously reported that early treatment with the anti-inflammatory cytokine interleukin 10 (IL-10) delays the onset of irreversible shock, defined as the time at which rescue surgery to remove the necrotic cecum is no longer effective. Because IL-10 can be immunostimulatory for T cells, we hypothesized that in the CLP model, late IL-10 treatment after removal of the infectious nidus at the onset of irreversible shock would restore T cell responsiveness and increase survival. C57BL/6J mice were subjected to lethal CLP with and without rescue surgery, concurrent with IL-10 treatment, at the onset of irreversible shock. Survival and serum IL-6 levels were measured as markers of the response to treatment. Ten hours after intervention, all groups exhibited T cell hyporesponsiveness marked by impaired interferon (IFN)-γ production by Con A-stimulated splenocytes. IL-6 levels at 10 h were related to outcome independent of treatment. By 25 h after intervention, only the dual treatment group of cecal removal and IL-10 exhibited T cell responsiveness that was similar to pre-CLP levels (P = 0.26) and had a 7-day survival of 90% (P ≤ 0.002 compared with all other groups). Thus, even in the advanced stages of septic shock when standard therapies fail, treatment with IL-10 extends the therapeutic window. For an individual mouse, the efficacy of such treatment may be predicted by an early postintervention IL-6 level.