No abstract

CD11b+Gr-1+ myeloid suppressor cells (MSC) accumulate in lymphoid organs under conditions of intense immune stress where they inhibit T and B cell function. We recently described the generation of immortalized MSC lines that provide a homogeneous source of suppressor cells for dissecting the mechanism of suppression. In this study we show that the MSC lines potently block in vitro proliferation of T cells stimulated with either mitogen or antigenic peptide, with as few as 3% of MSC cells causing complete suppression. Inhibition of mitogenic and peptide-specific responses is not associated with a loss in IL-2 production or inability to up-modulate the early activation markers, CD69 and CD25, but results in direct impairment of the three IL-2R signaling pathways, as demonstrated by the lack of Janus kinase 3, STAT5, extracellular signal-regulated kinase, and Akt phosphorylation in response to IL-2. Suppression is mediated by and requires NO, which is secreted by MSC in response to signals from activated T cells, including IFN-γ and a contact-dependent stimulus. Experiments with inducible NO synthase knockout mice demonstrated that the inhibition of T cell proliferation by CD11b+Gr-1+ cells in the spleens of immunosuppressed mice is also dependent upon NO, indicating that the MSC lines accurately represent their normal counterparts. The distinctive capacity of MSC to generate suppressive signals when encountering activated T cells defines a specialized subset of myeloid cells that most likely serve a regulatory function during times of heightened immune activity.

A number of pathogens induce immature dendritic cells (iDC) to migrate to lymphoid organs where, as mature DC (mDC), they serve as efficient APC. We hypothesized that pathogen recognition by iDC is mediated by Toll-like receptors (TLRs), and asked which TLRs are expressed during the progression of monocytes to mDC. We first measured mRNA levels for TLRs 1–5 and MD2 (a protein required for TLR4 function) by Northern analysis. For most TLRs, message expression decreased severalfold as monocytes differentiated into iDC, but opposing this trend, TLR3 and MD2 showed marked increases during iDC formation. When iDC were induced to mature with LPS or TNF-α, expression of most TLRs transiently increased and then nearly disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the activation of IL-1 receptor-associated kinase, an early component in the TLR signaling pathway, strongly suggesting that LPS signals through a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding, was very low, corresponding to a few thousand molecules per cell in monocytes, and a few hundred or less in iDC. We conclude that TLRs are expressed in iDC and are involved in responses to at least one pathogen-derived substance, LPS. If TLR4 is solely responsible for LPS signaling in humans, as it is in mice, then its extremely low surface expression implies that it is a very efficient signal transducer in iDC.

No abstract

We previously demonstrated that a specialized subset of immature myeloid cells migrate to lymphoid organs as a result of tumor growth or immune stress, where they suppress B and T cell responses to Ags. Although NO was required for suppression of mitogen activation of T cells by myeloid suppressor cells (MSC), it was not required for suppression of allogenic responses. In this study, we describe a novel mechanism used by MSC to block T cell proliferation and CTL generation in response to alloantigen, which is mediated by the enzyme arginase 1 (Arg1). We show that Arg1 increases superoxide production in myeloid cells through a pathway that likely utilizes the reductase domain of inducible NO synthase (iNOS), and that superoxide is required for Arg1-dependent suppression of T cell function. Arg1 is induced by IL-4 in freshly isolated MSC or cloned MSC lines, and is therefore up-regulated by activated Th2, but not Th1, cells. In contrast, iNOS is induced by IFN-γ and Th1 cells. Because Arg1 and iNOS share l-arginine as a common substrate, our results indicate that l-arginine metabolism in myeloid cells is a potential target for selective intervention in reversing myeloid-induced dysfunction in tumor-bearing hosts.

In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.

Atopic diseases such as allergy and asthma are characterized by increases in Th2 cells and serum IgE antibodies. The binding of allergens to IgE on mast cells triggers the release of several mediators, of which histamine is the most prevalent. Here we show that histamine, together with a maturation signal, acts directly upon immature dendritic cells (iDCs), profoundly altering their T cell polarizing capacity. We demonstrate that iDCs express two active histamine receptors, H1 and H2. Histamine did not significantly affect the LPS-driven maturation of iDCs with regard to phenotypic changes or capacity to prime naive T cells, but it dramatically altered the repertoire of cytokines and chemokines secreted by mature DCs. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DCs polarized naive CD4 + T cells toward a Th2 phenotype, as compared with DCs that had matured in the absence of histamine. We propose that the Th2 cells favor IgE production, leading to increased histamine secretion by mast cells, thus creating a positive feedback loop that could contribute to the severity of atopic diseases.

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum͞cis Golgi with differentsized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substances in vivo.M ammalian cells respond to lipopolysaccharide (LPS) (1), a major component of the outer membrane of Gramnegative bacterial cell walls, by activating Toll-like receptor 4 (TLR4) (1-5). TLR4 was first described as a member of a family of type I integral membrane proteins that contain numerous leucine-rich motifs on their extracellular portions and an intracellular signaling domain homologous to that of the IL-1 receptor (6, 7). In response to LPS, TLR4 initiates a cascade of serine͞threonine kinases that eventually leads to the transcription of genes involved in inflammation (8, 9). To respond efficiently to LPS, TLR4 requires an accessory protein, MD-2 (10). MD-2 is a 20-30-kDa glycoprotein that was originally discovered by sequence homology with MD-1, a protein found associated with a B cell homologue of TLR4, RP105 (11,12). MD-1 binds to the extracellular domain of RP105 and enhances its surface expression. Similarly, MD-2 binds to the extracellular domains of both TLRs 2 and 4 and causes their surface expression levels to increase (10, 13). Photoaffinity labeling studies have recently shown that LPS binds directly to the TLR4͞MD-2 complex and that both molecules are in close proximity to the bound LPS (14).MD-2, which contains a leader sequence but lacks a transmembrane domain, is secreted into the medium by transfected kidney epithelial cells (15). However, the structure and function of secreted MD-2 (sMD-2) have not been defined, nor is it known whether MD-2 is released into the medium by normal cells. To examine the properties of sMD-2, we have stably transfected HEK293 cells with an epitope-tagged version of MD-2 and have examined the secreted protein in culture supernatants. Based on previous findings from this laboratory that immature dendritic cells (iDC) express elevated levels of MD-2 message and decreased levels of TLRs 2 and 4 relative to mon...