Interleukin-10 (IL-10), a pleiotropic cytokine that inhibits inflammatory and cell-mediated immune responses, is produced by a wide variety of cell types including T and B cells and monocytes/macrophages. Regulation of pro-and anti-inflammatory cytokines has been suggested to involve distinct signaling pathways. In this study, we investigated the regulation of the human IL-10 (hIL-10) promoter in the human monocytic cell line THP-1 following activation with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences revealed that DNA sequences located between base pairs ؊652 and ؊571 are necessary for IL-10 transcription. A computer analysis of the promoter sequence between base pairs ؊652 and ؊571 revealed the existence of consensus sequences for Sp1, PEA1, YY1, and EpsteinBarr virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors. THP-1 cells transfected with a plasmid containing mutant Sp1 abrogated the promoter activity, whereas plasmids containing the sequences for PEA1, YY1, and EBNA-2-like transcription factors did not influence hIL-10 promoter activity. To understand the events upstream of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogenactivated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinases, failed to modulate IL-10 production. Furthermore, SB203580 inhibited LPS-induced activation of Sp1, as well as the promoter activity in cells transfected with a plasmid containing the Sp1 consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn regulates transcription of the hIL-10 gene.

SummaryMutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a C0014-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (me) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the me°mutation creates an insertion or deletion in the phosphatase domain. No PTPIC RNA or protein could be detected in the hemopoietic tissues ofme mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe me mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of me°PTPIC bound normally to phosphotyrosine-containing ligands in vitro . Nevertheless, the me mutation induced a marked reduction in PTPIC activity. These observations provide strong evidence that the motheaten phenotypic results from loss-offunction mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor-and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase ( The pivotal role of the SH2 domain-containing SHP-1 (PTP1C, HCP, or SHPTP1) tyrosine phosphatase in the regulation of hemopoietic cell growth and development is now well recognized (1). In contrast to the structurally similar, ubiquitously expressed SHP-2 (Syp or PTP1D) tyrosine phosphatase and its Drosophila csw homolog (10, 37), SHP-1 appears to exert primarily inhibitory effects on the signaling cascades in which it participates (34). SHP-1 has been shown, for example, to suppress the growth-promoting properties of the activated interleukin 3 (IL-3), erythropoietin, and colony-stimulating factor 1 receptors, an effect mediated either directly by receptor dephosphorylation or indirectly by dephosphorylation of receptor-associated protein tyrosine kinases (PTKs) (4,19,55,56). SHP-1 has also been implicated in downregulation of the signaling pathways evoked by engagement of the B-and Tlymphocyte antigen receptors (5, 32, 33), antigen receptor comodulators such as CD22, Fc␥RIIB, and CD5, and cytosolic signaling molecules such as Vav and Grb2/Sos1 which are involved in Ras activation (6,8,20). The capacity of SHP-1 to negatively modulate this broad spectrum of signaling effectors is consistent with the enormous overexpansion of multiple hemopoietic cell populations manifested by motheaten (me) and viable motheaten (me v ) mice, animals now known to be homozygous for loss-of-function mutations in the SHP-1 gene (21,46). The presence of two SH2 domains in SHP-1, as well as the possibility for altering its C-terminal SH2 domain by alternative splicing of a 39-amino-acid segment (46), provides a structural explanation for the diverse range of molecular interactions in which this phosphotyrosine phosphatase (PTP) appears to participate. Thus, while the precise structural basis for and physiologic effects of SHP-1 interactions with the molecules with which it has been shown to associate require further investigation, the current data concerning SHP-1 functions identify this PTP as a critical player in the modulation of hemopoietic cell growth and function.In addition to the regulation of cell proliferation, SHP-1 has also been implicated in the control of signaling cascades coupling growth factor receptors to hemopoietic cell differentiation. This role for SHP-1 has become particularly well appreciated in the context of data derived from studies of SHP-1 interactions with the transmembrane PTK receptor encoded by the c-Kit proto-oncogene. The latter receptor subserves pivotal functions in promoting the development, survival, and proliferation of hemopoietic stem cells, neural crest-derived cells, and germ cells, a role...

IL-12 plays a critical role in the development of cell-mediated immune responses and in the pathogenesis of inflammatory and autoimmune disorders. Dexamethasone (DXM), an anti-inflammatory glucocorticoid, has been shown to inhibit IL-12p40 production in LPS-stimulated monocytic cells. In this study, we investigated the molecular mechanism by which DXM inhibits IL-12p40 production by studying the role of the mitogen-activated protein kinases (MAPKs), and the key transcription factors involved in human IL-12p40 production in LPS-stimulated monocytic cells. A role for c-Jun N-terminal kinase (JNK) MAPK in LPS-induced IL-12p40 regulation in a promonocytic THP-1/CD14 cell line was demonstrated by using specific inhibitors of JNK activation, SP600125 and a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase-1 mutant. To identify transcription factors regulating IL-12p40 gene transcription, extensive deletion analyses of the IL-12p40 promoter was performed. The results revealed the involvement of a sequence encompassing the AP-1-binding site, in addition to that of NF-κB. The role of AP-1 in IL-12p40 transcription was confirmed by using antisense c-fos and c-jun oligonucleotides. Studies conducted to understand the regulation of AP-1 and NF-κB activation by JNK MAPK revealed that both DXM and SP600125 inhibited IL-12p40 gene transcription by inhibiting the activation of AP-1 and NF-κB transcription factors as revealed by luciferase reporter and gel mobility shift assays. Taken together, our results suggest that DXM may inhibit IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-κB transcription factors.

Inoculation of canola (Brassica campestris) seeds with a nitrogen-fixing strain of Pseudomonas putida (GR12-2) drastically increased the root length of seedlings grown in sterile growth pouches. Seed inoculation with inactive bacteria did not affect root lengths. Root elongation capacity was retained by nonnitrogen-fixing mutants of strain GR12-2. On the other hand, two other wild-type pseudomonads that do not fix nitrogen also increased root elongation. The addition of mineral nitrogen to the growth solution at concentrations of 1 mM or higher significantly inhibited root elongation of either inoculated or noninoculated seedlings. On the other hand, the addition of phosphate to the growth solution at similar concentrations stimulated root elongation of inoculated and noninoculated seedlings. The combined effects of bacterial inoculation and addition of phosphate on root and shoot elongation and on root and shoot weight were additive. Seed inoculation with P. putida GR12-2 increased the uptake of labelled phosphorus (32P) by seedlings grown in growth pouches and also enhanced the shoot elongation of seedlings grown in sterile soil. The capacity of P. putida GR12-2 to enhance phosphate uptake and to promote plant growth under gnotobiotic conditions may open the door to a new direction in the development of plant growth promoting inoculants.

SummaryRecent data implicating loss of PTPIC tyrosine phosphatase activity in the genesis of the multiple hemopoietic cell defects found in systemic autoimmune/immunodeficient motheaten (me) and viable motheaten (me ~) mice suggest that PTP1C plays an important role in modulating intracellular signaling events regulating cell activation and differentiation. To begin elucidating the role for this cytosolic phosphatase in lymphoid cell signal transduction, we have examined early signaling events and mitogenic responses induced by B cell antigen receptor (BCK) ligation in me and me ~ splenic B cells and in CD5 + CH12 lymphoma cells, which represent the lymphoid population amplified in motheaten mice. Despite their lack of functional Irl'PlC, me and me ~ B cells proliferated normally in response to LPS. However, compared with wild-type B cells, cells from the mutant mice were hyperresponsive to normally submitogenic concentrations of F(ab')2 anti-Ig antibody, and they exhibited reduced susceptibility to the inhibitory effects of Fc3'IIRB cross-linking on BCR-induced proliferation. Additional studies of unstimulated CH12 and wild-type splenic B cells revealed the constitutive association of PTPIC with the resting BCR complex, as evidenced by coprecipitation of PTPIC protein and phosphatase activity with BCR components and the depletion of BCK-associated tyrosine phosphatase activity by anti-PTPIC antibodies. These results suggest a role for trl'PlC in regulating the tyrosine phosphorylation state of the resting BCR complex components, a hypothesis supported by the observation that PTP1C specifically induces dephosphorylation of a 35-kD BCR-associated protein likely representing Ig-ot. In contrast, whereas membrane Ig cross-linking was associated with an increase in the tyrosine phosphorylation of PTP1C and an ,~140-kD coprecipitated protein, PTP1C was no longer detected in the BCR complex after receptor engagement, suggesting that PTP1C dissociates from the activated receptor complex. Together these results suggest a critical role for PTP1C in modulating BCR signaling capacity, and they indicate that the PTP1C influence on B cell signaling is likely to be realized in both resting and activated cells.

Chronic hepatitis B virus (HBV) infection is characterized by T cell tolerance to virus. Although inhibition of T cell responses by myeloid-derived suppressor cells (MDSCs) has been observed in patients with chronic hepatitis B (CHB), the mechanism for expansion of MDSCs remains ambiguous. In this study, a significant increased frequency of monocytic MDSCs (mMDSCs) was shown positively correlated to level of HBsAg in the patients with CHB. We further found hepatitis B surface Ag (HBsAg) efficiently promoted differentiation of mMDSCs in vitro, and monocytes in PBMCs performed as the progenitors. This required the activation of ERK/IL-6/STAT3 signaling feedback. Importantly, the mMDSCs polarized by HBsAg in vitro acquired the ability to suppress T cell activation. Additionally, treatment of all-trans retinoic acid, an MDSC-targeted drug, restored the proliferation and IFN-γ production by HBV-specific CD4+ and CD8+ T cells in PBMCs from patients with CHB and prevented increase of viral load in mouse model. In summary, HBsAg maintains HBV persistence and suppresses T cell responses by promoting differentiation of monocytes into mMDSCs. A therapy aimed at the abrogation of MDSCs may help to disrupt immune suppression in patients with CHB.

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