Ilkka Junttila

Tuning sensitivity to IL-4 and IL-13: differential expression of IL-4Rα, IL-13Rα1, and γc regulates relative cytokine sensitivity

Interleukin (IL)-4 and -13 are related cytokines sharing functional receptors. IL-4 signals through the type I (IL-4Rα/common γ-chain [γc]) and the type II (IL-4Rα/-13Rα1) IL-4 receptors, whereas IL-13 utilizes only the type II receptor. In this study, we show that mouse bone marrow–derived macrophages and human and mouse monocytes showed a much greater sensitivity to IL-4 than to IL-13. Lack of functional γc made these cells poorly responsive to IL-4, while retaining full responsiveness to IL-13. In mouse peritoneal macrophages, IL-4 potency exceeds that of IL-13, but lack of γc had only a modest effect on IL-4 signaling. In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts. Using levels of receptor chain expression and known binding affinities, we modeled the assemblage of functional type I and II receptor complexes. The differential expression of IL-4Rα, IL-13Rα1, and γc accounted for the distinct IL-4–IL-13 sensitivities of the various cell types. These findings provide an explanation for IL-13s principal function as an “effector” cytokine and IL-4s principal role as an “immunoregulatory” cytokine.

Type I IL-4Rs Selectively Activate IRS-2 to Induce Target Gene Expression in Macrophages

The ability of IL-4, but not IL-13, to efficiently activate IRS-2 leads to the expression of a subset of genes in alternatively activated macrophages. Selective Activation The T cell–derived cytokines interleukin-4 (IL-4) and IL-13 are closely related and are associated with allergic inflammatory responses, such as those that occur in asthma. IL-4 binds to the IL-4 receptor α (IL-4Rα) subunit, which heterodimerizes with either the γC subunit or the IL-13Rα1 subunit to form the type I or type II IL-4 receptors, respectively. Thus, whereas IL-4 activates both type I and type II receptors, IL-13 activates type II receptors. In allergic asthma, IL-4 is important in driving the differentiation of T helper type 2 (TH2) cells, whereas IL-13 plays a role in mucus secretion and airway hyperresponsiveness. Because of their overlap in receptor usage, it has been difficult to tease apart the different contributions of IL-4 and IL-13 (see the Perspective by Wills-Karp and Finkelman). By comparing the responses to each cytokine of a human cell line expressing type I and type II receptors to that of cells expressing only type II receptors, Heller et al. have found a critical difference in the signaling pathways activated by IL-4 and IL-13. Although both cytokines activated signal transducer and activator of transcription 6 to a similar extent, IL-4–stimulated phosphorylation and activation of insulin receptor substrate 2 (IRS-2) was much more efficient than that of IL-13 and was dependent on the presence of the γC subunit. Moreover, type I IL-4 receptor–mediated activation of IRS-2 resulted in the expression of a subset of genes associated with alternatively activated macrophages. By increasing our understanding of the differences between IL-4– and IL-13–dependent signaling pathways, these data may help in the development of more selective therapies against allergic asthma. Although interleukin-4 (IL-4) and IL-13 participate in allergic inflammation and share a receptor subunit (IL-4Rα), they have different functions. We compared cells expressing type I and II IL-4Rs with cells expressing only type II receptors for their responsiveness to these cytokines. IL-4 induced highly efficient, γC-dependent tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2), whereas IL-13 was less effective, even when phosphorylation of signal transducer and activator of transcription 6 (STAT6) was maximal. Only type I receptor, γC-dependent signaling induced efficient association of IRS-2 with the p85 subunit of phosphoinositide 3-kinase or the adaptor protein growth factor receptor–bound protein 2. In addition, IL-4 signaling through type I IL-4Rs induced more robust expression of a subset of genes associated with alternatively activated macrophages than did IL-13. Thus, IL-4 activates signaling pathways through type I IL-4Rs qualitatively differently from IL-13, which cooperate to induce optimal gene expression.

Sustained IL-4 exposure leads to a novel pathway for hemophagocytosis, inflammation, and tissue macrophage accumulation

Erythrophagocytosis and inflammation from activated macrophages occur in distinct clinical scenarios. The presence of CD8(+) T cells and interferon-γ (IFN-γ) production is required to induce disease in mouse models of hemophagocytic lymphohistiocytosis. We investigated the roles of a different class of proinflammatory cytokines, interleukin-4 (IL-4) and IL-13, in the induction of inflammatory tissue macrophage accumulation and/or hemophagocytosis. We found that large amounts of IL-4, but not IL-13, delivered via an implanted mini-pump or IL-4/anti-IL-4 complexes, lead to substantial YM1(+) tissue macrophage accumulation, erythrophagocytosis within the liver, spleen, and bone marrow, decreased hemoglobin and platelet levels, and acute weight loss. This effect is not dependent on the presence of antibody or T cells, as treatment of Rag2(-/-) mice leads to similar disease, and IFN-γ neutralization during IL-4 treatment had no effect. IL-4 treatment results in suppression of IL-12, elevation of serum IFN-γ, IL-10, and the murine IL-8 homolog KC, but not IL-6, IL-1β, or tumor necrosis factor-α. Finally, mice transgenic for IL-4 production developed tissue macrophage accumulation, disruption of splenic architecture, bone marrow hypocellularity, and extramedullary hematopoiesis. These data describe a novel pathophysiologic pathway for erythrophagocytosis in the context of tissue macrophage accumulation and inflammation involving elevations in IL-4 and alternative macrophage activation.

Efficient cytokine-induced IL-13 production by mast cells requires both IL-33 and IL-3

BACKGROUND IL-13 is a critical effector cytokine for allergic inflammation. It is produced by several cell types, including mast cells, basophils, and TH2 cells. In mast cells and basophils its induction can be stimulated by cross-linkage of immunoglobulin receptors or cytokines. The IL-1 family members IL-33 and IL-18 have been linked to induction of IL-13 production by mast cells and basophils. In CD4 TH2 cells IL-33-mediated production of IL-13 requires simultaneous signal transducer and activator of transcription (STAT) 5 activation. OBJECTIVE Here we have addressed whether cytokine-induced IL-13 production in mast cells and basophils follows the same logic as in TH2 cells: requirement of 2 separate signals. METHODS By generating a bacterial artificial chromosome (BAC) transgenic IL-13 reporter mouse, we measured IL-13 production in mast cells and basophils. RESULTS In mast cells harvested from peritoneal cavities, 2 cytokine signals are required for IL-13 production: IL-33 and IL-3. In bone marrow mast cells IL-13 production requires IL-33, but the requirement for a STAT5 inducer is difficult to evaluate because these cells require the continuous presence of IL-3 (a STAT5 activator) for survival. Poorer STAT5 inducers in culture (IL-4 or stem cell factor) result in less IL-13 production on IL-33 challenge, but the addition of exogenous IL-3 enhances IL-13 production. This implies that bone marrow-derived mast cells, like peritoneal mast cells and TH2 cells, require stimulation both by an IL-1 family member and a STAT5 inducer to secrete IL-13. Basophils follow the same rule; splenic basophils produce IL-13 in response to IL-18 or IL-33 plus IL-3. CONCLUSION Optimal IL-13 production from mast cells and basophils requires 2 cytokine signals.

TSLP Expression: Analysis with a ZsGreen TSLP Reporter Mouse

Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays a central role in induction of allergic inflammatory responses. Its principal targets have been reported to be dendritic cells and/or CD4 T cells; epithelial cells are a principal source. We report in this study the development of a reporter mouse (TSLP-ZsG) in which a ZsGreen (ZsG)-encoding construct has been inserted by recombineering into a bacterial artificial chromosome immediately at the translation initiating ATG of TSLP. The expression of ZsG by mice transgenic for the recombinant BAC appears to be a faithful surrogate for TSLP expression, particularly in keratinocytes and medullary thymic epithelial cells. Limited ZsG and TSLP mRNA was observed in bone marrow–derived mast cells, basophils, and dendritic cells. Using the TSLP-ZsG reporter mouse, we show that TNF-α and IL-4/IL-13 are potent inducers of TSLP expression by keratinocytes and that local activation of Th2 and Th1 cells induces keratinocyte TSLP expression. We suggest that the capacity of TSLP to both induce Th2 differentiation and to be induced by activated Th2 cells raises the possibility that TSLP may be involved in a positive feedback loop to enhance allergic inflammatory conditions.

NKG2D gene variation and susceptibility to viral bronchiolitis in childhood

BackgroundGenetic factors associated with bronchiolitis are inadequately characterized. We therefore inspected a selected subpopulation of our previous genome-wide association study (GWAS) of bronchiolitis for overlap with known quantitative trait loci (QTLs) to identify susceptibility loci that potentially affect mRNA and protein levels.MethodsGWAS included a Finnish–Swedish case–control population (n = 187), matched for age and site. We integrated GWAS variants (p < 10−4) with QTL data. We subsequently verified allele-specific expression of identified QTLs by flow cytometry. Association of the resulting candidate loci with bronchiolitis was tested in three additional cohorts from Finland and Denmark (n = 1201).ResultsBronchiolitis-susceptibility variant rs10772271 resided within QTLs previously associated with NKG2D (NK group 2, member D) mRNA and protein levels. Flow cytometric analysis confirmed the association with protein level in NK cells. The GWAS susceptibility allele (A) of rs10772271 (odds ratio [OR] = 2.34) corresponded with decreased NKG2D expression. The allele was nominally associated with bronchiolitis in one Finnish replicate (OR = 1.50), and the other showed directional consistency (OR = 1.43). No association was detected in Danish populationConclusionsThe bronchiolitis GWAS susceptibility allele was linked to decreased NKG2D expression in the QTL data and in our expression analysis. We propose that reduced NKG2D expression predisposes infants to severe bronchiolitis.