CBL-GRB2 interaction in myeloid immunoreceptor tyrosine activation motif signaling

In this study, we provide the first evidence for role of the CBL adapter protein interaction in Fc gammaRI receptor signal transduction. We study the Fc gammaRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-gamma-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc gammaRIgamma of Fc gammaRI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc gammaRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc gammaRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461-670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc gammaRI signaling.     

Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state

The receptor-associated protein tyrosine kinases JAK1 and JAK2 are both required for the interferon (IFN)-gamma response. The effects of expressing kinase-negative JAK mutant proteins on signal transduction in response to IFN-gamma in wild-type cells and in mutant cells lacking either JAK1 or JAK2 have been analysed. In cells lacking endogenous JAK1 the expression of a transfected kinase-negative JAK1 can sustain substantial IFN-gamma-inducible gene expression, consistent with a structural as well as an enzymic role for JAK1. Kinase-negative JAK2, expressed in cells lacking endogenous JAK2, cannot sustain IFN-gamma-inducible gene expression, despite low level activation of STAT1 DNA binding activity. When expressed in wild-type cells, kinase-negative JAK2 acts as a dominant-negative inhibitor of the IFN-gamma response. Further analysis of the JAK/STAT pathway suggests a model for the IFN-gamma response in which the initial phosphorylation of JAK1 and JAK2 is mediated by JAK2, whereas phosphorylation of the IFN-gamma receptor is normally carried out by JAK1. The efficient phosphorylation of STAT 1 in the receptor-JAK complex may again depend on JAK2. Interestingly, a JAK1-dependent signal, in addition to STAT1 activation, appears to be required for the expression of the antiviral state.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function

Regulation of leukocyte integrin avidity is a crucial aspect of inflammation and immunity. The actin cytoskeleton has an important role in the regulation of integrin function, but the cytoskeletal proteins involved are largely unknown. Because inflammatory stimuli that activate integrin-mediated adhesion in human polymorphonuclear neutrophils (PMN) and monocytes cause phosphorylation of the actin-bundling protein L-plastin, we tested whether L-plastin phosphorylation was involved in integrin activation. L-plastin-derived peptides that included the phosphorylation site (Ser-5) rapidly induced leukocyte integrin-mediated adhesion when introduced into the cytosol of freshly isolated primary human PMN and monocytes. Substitution of Ala for Ser-5 abolished the ability of the peptide to induce adhesion. Peptide-induced adhesion was sensitive to pharmacologic inhibition of phosphoinositol 3-kinase and protein kinase C, but adhesion induced by a peptide containing a phosphoserine at position 5 was insensitive to inhibition. These data establish a novel role for L-plastin in the regulation of leukocyte adhesion and suggest that many signaling events implicated in integrin regulation act via induction of L-plastin phosphorylation.     

The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes

The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and -2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcgamma receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor gamma chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.     

Eosinophil recruitment to the lung in a murine model of allergic inflammation. The role of T cells, chemokines, and adhesion receptors

Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.     

A 16-element phased-array head coil

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.     

Integrin-mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signaling pathways in fibroblasts

Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2-Sos complex. Since Grb2-Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a beta1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.     

Prolactin receptor heterogeneity in bovine fetal and maternal tissues

Recombinant human interferon-gamma (rhIFN-gamma) decreases the frequency of serious infections in patients with chronic granulomatous disease (CGD) through an unknown mechanism. To test the hypothesis that it exerts a beneficial effect by enhancing clearance of microbes from the bloodstream and tissues, normal human subjects were treated in vivo with rhIFN-gamma. Phagocyte opsonic receptor expression, serum opsonin levels, and phagocytosis of bacteria were then measured. A 4.7-fold increase in neutrophil expression of the high-affinity Fc gamma-receptor (Fc gammaRI) was observed that peaked 48 hours after the initiation of rhIFN-gamma treatment (P < .05). Monocyte expression of Fc gammaRI, Fc gammaRII, Fc gammaRIII, CD11a, CD11b, CD18, and HLA-DR also significantly increased with peak expression at 48 hours. Phagocytosis by neutrophils of killed Staphylococcus aureus opsonized with heat-inactivated pooled human serum significantly improved after rhIFN-gamma treatment (P < .05) and correlated with Fc gammaRI expression by neutrophils (r = .8, P < .001). This increase in ingestion could be inhibited by anti-Fc gammaRI monoclonal antibodies. Levels of the serum opsonin lipopolysaccharide-binding protein also significantly increased after in vivo rhIFN-gamma (P < .05). These results suggest that the protective effect of rhIFN-gamma in patients with CGD may involve improved microbial clearance. Moreover, improved phagocyte trafficking may occur secondary to increased expression of monocyte beta2-integrins. Because these IFN-gamma-related improvements in host defense were seen in normal hosts, rhIFN-gamma may have broader applications in the treatment of various disorders of immunity in addition to its demonstrated efficacy in CGD.     

Utilization of health care services by immigrants and other ethnic/cultural groups in Ontario

Blood-contacting biomaterials may activate the complement cascade, thus promoting leukocyte adhesion to the biomaterial surface. We hypothesize that the extent of complement activation is modulated by biomaterial formulation and the presence of fluid shear stress. To investigate this hypothesis, we tested base poly(etherurethane ureas) formulated with or without Santowhite antioxidant, a nucleophilic additive. We found that adherent leukocyte densities decreased with increasing shear stress. Moreover, leukocyte adhesion was decreased significantly further by Santowhite additive under shear stress but not under static conditions. Monocytes showed a higher propensity for adhesion than did neutrophils under shear and static conditions. Under static conditions, adherent cells on the Santowhite-containing polyurethane had a slightly more activated morphology than those on the base polyurethane. Cell adhesion under shear stress was significantly decreased when C3 or fibronectin was depleted from the suspension medium. Santowhite additive increased Factor B adsorption to the test surface while shear stress increased Factor H adsorption. The combination of Santowhite additive and shear stress increased the adsorption of both Factor B and Factor H and the serum protein S-terminal complement complex levels, but it did not further increase the state of activation of adherent cells. We conclude that leukocyte adhesion on poly(etherurethane urea) surfaces is sensitive to the levels of shear stress and that both C3 and fibronectin are required to maintain adhesion in the presence of shear stress. The low state of cellular activation and increased Factor H adsorption may explain the decreased adherent leukocyte density on the Santowhite-containing polyurethane.     

Identification of a domain on the integrin alpha5 subunit implicated in cell spreading and signaling

The alpha5 beta1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha5 beta1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells, which are deficient in alpha5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha5 beta1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.     

Experimental studies on bacterial product CANTASTIM derived from Pseudomonas aeruginosa with immunomodulatory properties. IV. Tyrosine phosphorylation as an effect of stimulation on different cell populations

The bacterial product derived from Pseudomonas aeruginosa (trade mark-CANTASTIM) proved immunomodulatory effects in different systems, both in vitro and in vivo experimental animal models, as well as in clinical trials. Among the results obtained regarding CANTASTIM, the following immunomodulatory properties could be mentioned: an increase of the activated T cell subpopulations and humoral-mediated immune processes, facilitation of phagocytic processes, stimulation of cytotoxic activity reflected in the improvement of the capacity of defense in several tumoral and infectious diseases. To better elucidate the intimate mechanisms by which CANTASTIM modulates the cellular functions on different cellular populations, we used tyrosine phosphorylation as an estimate of cell activation on peripheral blood lymphocytes (PBL) and a monocyte cell line (THP-1). In PBL, the treatment with CANTASTIM renders them more susceptible to CD3 stimulation than non-treated cells. In monocytes, CANTASTIM and two phospholipid components of CANTASTIM modulated in a different manner the cellular adhesion on fibronectin and tyrosine phosphorylation leading to the conclusion that these phospholipid components do not fully explain CANTASTIM modulatory properties on cell adhesion processes.     

Molecular fine-specificity analysis of antibody responses to human cytomegalovirus and design of novel synthetic-peptide-based serodiagnostic assays

BACKGROUND: Glomerular monocyte infiltration is an early feature of lipid-mediated renal injury in animal models. Interactions between mesangial and infiltrating mononuclear cells may contribute to the development of glomerular scarring. METHODS: Adherence of U-937 monocytes to low-density lipoprotein (LDL)- or tumor necrosis factor alpha (TNFalpha)-prestimulated human mesangial cells was assessed by colorimetry of nuclear staining with crystal violet. Blocking antibodies were added to examine the mechanisms of binding. Adhesion molecule expression and fibronectin synthesis were measured by ELISA. RESULTS: Preincubation of mesangial cells for 24 hours with LDL (100 micrograms/ml) or mildly oxidized (minimally modified) LDL (MM-LDL) increased monocyte adhesion by 207% and 240%, respectively, compared with control nonstimulated cells (100%). TNFalpha (100 U/ml) enhanced binding by 335% and up-regulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by 505% and 179%, respectively, as compared with MM-LDL (120% and 116%) and LDL, which had no effect. Blocking antibodies to these adhesion molecules inhibited monocyte binding to TNFalpha- and, to a lesser extent, MM-LDL-primed mesangial cells, but had no effect after LDL pretreatment. In contrast to TNFalpha, MM-LDL and LDL increased mesangial cell-associated fibronectin, whereas antibodies to fibronectin inhibited monocyte binding to lipoprotein-stimulated but not TNFalpha-stimulated cells. CONCLUSIONS: Although enhanced monocyte adhesion to TNFalpha- and, to a lesser extent, MM-LDL-stimulated mesangial cells is mediated by changes in ICAM-1 and VCAM-1 expression, both LDL and MM-LDL promote similar cellular interactions as a result of increased fibronectin production.