Person:

Welch, John

The diverse functions of macrophages as participants in innate and acquired immune responses are regulated by the specific milieu of environmental factors, cytokines, and other signaling molecules that are encountered at sites of inflammation. Microarray analysis of the transcriptional response of mouse peritoneal macrophages to the TH2 cytokine interleukin-4 (IL-4) identified Ym1 and arginase as the most highly up-regulated genes, exhibiting more than 68- and 88-fold induction, respectively. Molecular characterization of the Ym1 promoter in transfected epithelial and macrophage cell lines revealed the presence of multiple signal transducers and activators of transcription 6 (STAT6) response elements that function in a combinatorial manner to mediate transcriptional responses to IL-4. The participation of STAT6 as an obligate component of protein complexes binding to these sites was established by analysis of nuclear extracts derived from STAT6-deficient macrophages. Macrophage expression of Ym1 was highly induced in vivo by an IL-4- and STAT6-dependent mechanism during the evolution of allergic peritonitis, supporting the biological relevance of the IL-4-dependent pathway characterized ex vivo in peritoneal macrophages. These studies establish Ym1 as a highly inducible STAT6-dependent transcript in TH2-biased inflammation and define Cis-active elements in the Ym1 promoter that are required for this transcriptional response.

The macrophage plays an important role bridging between innate and acquired immune function. It is capable both of responding to nonspecific stimuli, such as bacterial lipopolysaccarides (LPS)1 and activated complement factors, but can also engage processes of the acquired immune response such as antibody-dependent opsonization and subsequent antigen presentation to T-cells (1, 2).

Macrophage function can be influenced profoundly by cytokines released from T-cells and other immune cells. These cytokines have been divided largely along two axes: TH1 cytokines, dominated by INF-γ and TH2 cytokines, dominated by IL-4 (3). Macrophage stimulation by TH1 cytokines results in free-radical release and increased cytokine secretion, implicated as essential signaling components of a successful response to infection by intracellular bacteria and viruses (4-6). In contrast, efficient defense against extracellular pathogens and parasites requires a different response, characterized by the release of TH2 cytokines (7). These mediate B-cell class switching to IgE antibodies, macrophage opsonization of soluble antigens, and activation of mast cells, basophils, and eosinophils. These two cytokine axes have been proposed to be mutually antagonistic, both in the commitment of T-cells toward a TH1 or TH2 lineage and in their influence on effector cells such as the macrophage.

In parallel with the separation of T-cells into either TH1- or TH2-biased cells, a number of groups have proposed recently that the exposure of macrophages to a specific set of cytokines also biases them toward either an M-1 phenotype (activated macrophages) or an M-2 phenotype (alternatively activated macrophages) (8-10). Macrophage exposure to IL-4 substantially blunts subsequent LPS- or INFγ-stimulated production of cytokines (IL-6, IL-8, IL-12, and tumor necrosis factor-α), receptors (CD14, FcγI, FcγII, and FcγIII), and superoxide radicals (11-14). At the same time, macrophage exposure to IL-4 results in the increased expression of scavenger receptors (the mannose receptor, the scavenger receptor type I, and CD163), CD13 (the aminopeptidase, capable of inactivating inflammatory mediators), and CD23 (FcεRII), thus blunting the cytotoxic effects of the macrophage and shifting its opsonizing focus from IgG antibodies to IgE while increasing its phagocytic capabilities (9, 15).

Allergic challenge of BALB/c mice with ovalbumin following sensitization has been shown to result in the recruitment of macrophages into an environment enriched in IL-4 and IL-5 (16-18). This allergic response provides a useful model of macrophages activated alternatively because of the relative abundance of macrophages, the cytokine milieu they are recruited into, and their high expression of arginase I, a marker of macrophages activated alternatively (19). In addition, this model of allergic response has been used to characterize general allergic phenomenon as they may apply to human pathophysiology of allergy and asthma.

INF-γ and LPS induce large and profound transcriptional changes in the macrophage (13, 20, 21). In contrast, relatively few genes have been characterized as direct targets of IL-4 stimulation. The development of large scale expression arrays allows near global glimpses of changes in gene expression profiles during cell proliferation, division, and in response to environmental signals. We applied this technology to probe the transcriptional response of murine macrophages to the quintessential TH2 cytokine, IL-4. Ym1 was one of the most highly induced IL-4 target genes, exhibiting a level of induction of 70-fold or greater in multiple macrophage populations. Studies of the Ym1 promoter suggest that the binding of STAT6 to multiple sites within a 600-bp region up-stream of the transcriptional start site is required for the IL-4 response in BEAS-2B cells and RAW macrophages. Ym1 was also highly induced in macrophages recruited to the peritoneal cavity during allergic peritonitis in an IL-4- and STAT6-dependent manner. Collectively, these findings establish molecular mechanisms underlying the transcriptional response of the most highly induced IL-4 target gene identified to date in macrophages.