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Signaling lymphocytic activation molecule-associated protein (SAP), an adaptor molecule that recruits Fyn to the signaling lymphocytic activation molecule (SLAM) family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4(+) T cells from SAP-deficient mice have defective TCR-induced and follicular Th cell IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4(+) T cells exhibit decreased protein kinase C (PKC)-theta recruitment upon TCR stimulation. We demonstrate in this paper using GST pulldowns and coimmunoprecipitation studies that SAP constitutively associates with PKC- in T cells. SAP-PKC-theta interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP's interactions with PKC-theta occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-theta recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-theta, like SAP, was required for SLAM-mediated increases in IL-4 production, and, conversely, membrane-targeted PKC-theta mutants rescued IL-4 expression in SAP(-/-) CD4(+) T cells, providing genetic evidence that PKC-theta is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production.

Dendritic cell (DC) apoptosis has been shown to play a role in maintaining a balance between tolerance and immunity. However, the mechanisms of how DC apoptosis affects the immune response are unclear. We have shown that in vitro culture of apoptotic DCs with immature DCs, results in their uptake by immature DCs, which subsequently turn into tolerogenic DCs, which then secrete TGF-beta1 and induce Foxp3(+) regulatory T cells (T(regs)). In this study we looked at the effects of apoptotic DCs in vivo. Here we show that apoptotic DCs are taken up by viable DCs in vivo, which suppresses the ability of viable DCs to undergo maturation and subsequent migration to the lymph nodes in response to LPS. Additionally, delivery of apoptotic DCs to LPS inflamed lungs results in resolution of inflammation, which is mediated by the ability of apoptotic DCs to suppress response of viable DCs to LPS. Additionally, apoptotic DCs also induce TGF-beta1 secretion in the mediastinal lymph nodes, which results in expansion of Foxp3(+) T(regs). Most importantly, we show that delivery of apoptotic DCs followed by OVA in CFA to mice suppresses T cell response to OVA and instead induces de novo generation of OVA-specific T(regs). Furthermore, delivery of apoptotic DCs followed by OVA in CFA results in expansion of T(regs) in TCR transgenic (OT-II) mice. These findings demonstrate that apoptotic DCs are taken up by viable DCs in vivo, which promotes tolerance through suppression of DC maturation and induction of T(regs).

Neutrophil chemotaxis depends on actin dynamics, but the roles for specific cytoskeleton regulators in this response remain unclear. By analysis of mammalian diaphanous-related formin 1 (mDia1)-deficient mice, we have identified an essential role for this actin nucleator in neutrophil chemotaxis. Lack of mDia1 was associated with defects in chemoattractant-induced neutrophil actin polymerization, polarization, and directional migration, and also with impaired activation of RhoA, its downstream target p160-Rho-associated coil-containing protein kinase (ROCK), and the leukemia-associated RhoA guanine nucleotide exchange factor (LARG). Our data also revealed mDia1 to be associated with another cytoskeletal regulator, Wiskott-Aldrich syndrome protein (WASp), at the leading edge of chemotaxing neutrophils and revealed polarized morphology and chemotaxis to be more mildly impaired in WAS(-/-) than in mDia1(-/-) neutrophils, but essentially abrogated by combined mDia1/WASp deficiency. Thus, mDia1 roles in neutrophil chemotaxis appear to be subserved in concert with WASp and are realized at least in part by activation of the LARG/RhoA/ROCK signaling pathway.

The mechanism of cytokine secretion is not well understood, but cytokines appear to be synthesized and released in a polarized fashion toward an Ag-specific target cell. In this study, we demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an essential component of the cytokine secretory pathway in CD4(+) T cells. Murine WASp-deficient CD4(+) T cells fail to polarize cytokines toward a target and show an unexpected and striking block in cytokine secretion. In contrast, chemokine secretion and trafficking of plasma membrane proteins, transported via the constitutive secretory pathway, are unaffected by the lack of WASp. These results suggest that CD4(+) T cell cytokines require a specialized, WASp-dependent pathway for cellular traffic and/or vesicle release that is distinct from that required for chemokine release. We propose that the use of different secretory pathways for cytokines and chemokines enables CD4(+) T cell activity to be further fine-tuned to serve specialized effector functions.

Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and me(v)/me(v) mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. MOTHEATEN: neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.

Restimulation of Ag receptors on peripheral T lymphocytes induces tyrosine phosphorylation-based signaling cascades that evoke Fas ligand expression and induction of Fas-mediated programmed cell death. In view of the role for the Src homology domain 2-bearing protein tyrosine phosphatase-1 (SHP-1) in modulating TCR signaling, we investigated the influence of SHP-1 on TCR-mediated apoptosis by assaying the sensitivity of peripheral T cells from SHP-1-deficient viable motheaten (mev) mice to cell death following TCR restimulation. The results of these studies revealed mev peripheral T cells to be markedly more sensitive than wild-type cells to induction of cell death following TCR stimulation. By contrast, PMA/ionophore and anti-Fas Ab-induced apoptotic responses were no different in mev compared with wild-type activated cells. Enhanced apoptosis of TCR-restimulated mev lymphocytes was associated with marked increases in Fas ligand expression as compared with wild-type cells, but was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Thus, the increased sensitivity of mev T cells to apoptosis following TCR restimulation appears to reflect a TCR-driven phenomenon mediated through up-regulation of Fas-Fas ligand interaction and induction of the Fas signaling cascade. These findings, together with the hyperproliferative responses of mev peripheral T cells to initial TCR stimulation, indicate that SHP-1 modulation of TCR signaling translates to the inhibition of both T cell proliferation and activation and, as such, is likely to play a pivotal role in regulating the expansion of Ag-stimulated T cells during an immune response.

The SH2 domain containing tyrosine phosphatase SHP-1 has been implicated in the regulation of a multiplicity of signaling pathways involved in hemopoietic cell growth, differentiation, and activation. A pivotal contribution of SHP-1 in the modulation of myeloid cell signaling cascades has been revealed by the demonstration that SHP-1 gene mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten mice. To investigate the role of SHP-1 in regulation of myeloid leukocytes, an HA epitope-tagged dominant negative (interfering) SHP-1 (SHP-1C453S) was expressed in the myelo-monocytic cell line U937 using the pcDNA3 vector. Overexpression of this protein in SHP-1C453S transfectants was demonstrated by Western blot analysis and by detection of decreased specific activity. Growth, proliferation, and IL-3-induced proliferative responses were substantially increased in the SHP-1C453S-overexpressing cells relative to those in control cells. The results of cell cycle analysis also revealed that the proportion of cells overexpressing SHP-1C453S in S phase was greater than that of control cells. The SHP-1C453S-expressing cells also displayed diminished rates of apoptosis as detected by flow cytometric analysis of propidium iodide-stained cells and terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end-labeling assay. While motility and phagocytosis were not affected by SHP-1C453S overexpression, adhesion and the oxidative burst in response to PMA were enhanced in the SHP-1C453S compared with those in the vector alone transfectants. Taken together, these results suggest that SHP-1 exerts an important negative regulatory influence on cell proliferation and activation while promoting spontaneous cell death in myeloid cells.

Platelet-activating factor (PAF) may play a role in the regulation of immune responsiveness and is a potent mediator in asthmatic inflammation. However, as yet, the mechanisms whereby PAF mediates its pleiomorphic effects on immune cells have not been elucidated. Because PAF is a potent chemotactic factor for eosinophils, the presence of receptors for PAF (PAFR) on lymphocytes may provide a mechanism for the concurrent recruitment of both eosinophils and T lymphocytes into the airways of asthmatic patients. To address this issue, we have examined freshly isolated PBMC and granulocytic cells as well as various T and B lymphocyte lines with regards to PAFR expression and PAF-induced changes in intracellular calcium concentration. Using two-color immunofluorescence techniques and highly purified cell populations, it was not possible to detect surface PAFR protein or functional PAFR on resting and in vivo or in vitro activated T and B cells derived from nonallergic individuals or patients with allergic asthma. In addition, we were unable to detect PAFR mRNA, protein, or functional response to PAF in human or murine T cell lines. In contrast, we found functional PAFR in most B lymphoblastoid cell lines. Within the PBMC population, CD14+ cells respond to PAF. These results suggest that PAF does not interact directly with lymphocytes and thus that previous observations suggestive of such an interaction likely reflect the effects of PAF on monocytes. PAF-induced increases in intracellular calcium concentration were also detected in neutrophils and eosinophils, but were lower in granulocytes relative to the levels detected in monocytes.

In order to investigate the genetic basis for natural anti-DNA immune responses, we isolated and sequenced the variable gene elements (VH and VL) encoding an anti-DNA antibody expressed by a human hybridoma of normal origin (Kim4.6) and compared these sequences with those reported for four other human anti-DNA antibodies. The Kim4.6 antibody leader and VH segments were identical in nucleotide sequence with the VH1.9III germ-line VH3 gene, and the Kim4.6VL segment showed 98% nucleotide sequence identity with a V lambda I subgroup gene expressed in a Burkitt's lymphoma. Comparative analysis of Kim4.6 and other human hybridoma anti-DNA antibodies indicated that anti-DNA immune responses are diverse in terms of VH and VL gene utilization but may exhibit a bias toward rearrangement of VH genes that are over-represented in the fetal pre-B cell repertoire. Moreover, Kim4.6 and three of four other sequenced human anti-DNA antibodies appear to use a germ-line diversity gene, DXP'1, which may represent a counterpart of the DFL16.1 segment utilized in murine responses to the hapten nitrophenyl. Taken together, our findings indicate that anti-DNA immune responses can be encoded by nonmutated VH genes and that the elements and molecular mechanisms which engender this response are essentially the same among natural and lupus-associated anti-DNA antibodies. Our data also suggest that natural autoimmune responses originate early in B cell ontogeny as is consistent with the hypothesis that autoreactivity plays a major role in shaping the normal immune repertoire.

Autoantibodies that bind aminoacyl-tRNA synthetases are strongly associated with the human inflammatory myopathies polymyositis and dermatomyositis, but their molecular origins and relationship to pathogenesis are not known. To address these issues, we wished to identify the autoantigenic epitopes which react with these autoantibodies and to this end, we previously isolated a full length cDNA clone encoding the target Ag recognized most frequently by myositis sera, histidyl-tRNA synthetase (HRS). In the present study, we have analyzed the HRS autoepitopes by two amino acid insertion linker mutagenesis of HRS proteins expressed in Cos 1 cells. A series of mutant HRS cDNA were constructed and the expressed proteins were tested for enzyme activity and for immune reactivity with a panel of sera with anti-Jo-1 antibodies. Immunoblotting and immunoprecipitation analyses revealed that anti-Jo-1 antibodies recognize multiple conformation-dependent and independent epitopes on HRS and that the autoepitopes vary among different myositis patients.