Biomedical subjects

Jordan S Pober

Publications and source records attributed to Jordan S Pober.

At least 37 records · Page 2Linked to original sources

PubMed2005

Endothelial cells promote human immunodeficiency virus replication in nondividing memory T cells via Nef-, Vpr-, and T-cell receptor-dependent activation of NFAT.

Human endothelial cells (ECs) enhance human immunodeficiency virus (HIV) replication within CD4(+) memory T cells by 50,000-fold in a Nef-dependent manner. Here, we report that EC-mediated HIV type 1 replication is also dependent on an intact vpr gene. Moreover, we demonstrate that despite a requirement for engaging major histocompatibility complex (MHC) class II molecules and costimulators, EC-stimulated virus-producing cells (p24(high) T cells) do not proliferate, nor are they arrested in the cell cycle. Rather, they are minimally activated, sometimes expressing CD69 but not CD25, HLA-DR, VLA-1, or effector cytokines. Blocking antibodies to interleukin 2 (IL-2), IL-6, IL-7, or tumor necrosis factor do not inhibit viral replication. Cyclosporine effectively inhibits viral replication, as does disruption of the NFAT binding site in the viral long terminal repeat. Furthermore, in the presence of ECs, suboptimal T-cell receptor (TCR) stimulation with phytohemagglutinin L supports efficient viral replication, and suboptimal stimulation with toxic shock syndrome toxin 1 leads to viral replication selectively in the TCR-stimulated, Vbeta2-expressing T cells. Collectively, these data indicate that ECs provide signals that promote Nef- and Vpr-dependent HIV replication in memory T cells that have been minimally activated through their TCRs. Our studies suggest a mechanism for HIV replication in vivo within the reservoir of circulating memory CD4(+) T cells that persist despite antiretroviral therapy and further suggest that maintenance of immunological memory by MHC class II-expressing ECs via TCR signaling may contribute to HIV rebound following cessation of antiretroviral therapy.

Antigens, CD↗

PubMed2004

Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells.

Implants of collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVECs) induce the formation of human endothelial cell (EC)/murine vascular smooth muscle cell (VSMC) chimeric vessels in immunodeficient mice. Microfil casting of the vasculature 60 d after implantation reveals highly branched microvascular networks within the implants that connect with and induce remodeling of conduit vessels arising from the abdominal wall circulation. Approximately 85% of vessels within the implants are lined by Bcl-2-positive human ECs expressing VEGFR1, VEGFR2, and Tie-2, but not integrin alpha(v)beta(3). The human ECs are seated on a well formed human laminin/collagen IV-positive basement membrane, and are surrounded by mouse VSMCs expressing SM-alpha actin, SM myosin, SM22alpha, and calponin, all markers of contractile function. Transmission electron microscopy identified well formed EC-EC junctions, chimeric arterioles with concentric layers of contractile VSMC, chimeric capillaries surrounded by pericytes, and chimeric venules. Bcl-2-HUVEC-lined vessels retain 70-kDa FITC-dextran, but not 3-kDa dextran; local histamine rapidly induces leak of 70-kDa FITC-dextran or India ink. As in skin, TNF induces E-selectin and vascular cell adhesion molecule 1 only on venular ECs, whereas intercellular adhesion molecule-1 is up-regulated on all human ECs. Bcl-2-HUVEC implants are able to engraft within and increase perfusion of ischemic mouse gastrocnemius muscle after femoral artery ligation. These studies show that cultured Bcl-2-HUVECs can differentiate into arterial, venular, and capillary-like ECs when implanted in vivo, and induce arteriogenic remodeling of the local mouse vessels. Our results support the utility of differentiated EC transplantation to treat tissue ischemia.

Animals↗

PubMed2004

Transfer of human leukocytes into double-knockout Pfp-/-Rag2-/- mice grafted with human skin: increased accumulation of neutrophils in human dermal microvessels.

Severe combined immunodeficient mice reconstituted with human leukocytes have been useful to model parts of the human immune system, including some of its diseases (e.g., AIDS). Because no human polymorphonuclear leukocytes (huPMN) develop in these xenograft models, diseases such as several forms of vasculitis cannot be modeled using this approach. To provide such a model for vasculitis, human skin patches were grafted onto double-knockout Pfp(-/-)Rag2(-/-) mice, which not only lack functional T and B cells but which are also devoid of natural killer cells. After intravenous injection, a high proportion of huPMNs survived within the circulation and accumulated in the human blood vessels. The accumulation increased considerably after the endothelium of the skin patches had been stimulated by tumor necrosis factor-alpha. Alpha mild perivascular neutrophilic infiltration and vascular necrosis was observed in the microvessels of the skin patches. Thus, a xenograft model of vasculitis with predominant huPMNs infiltration has been established for the first time.

Animals↗

PubMed2004

Bcl-2 transduction protects human endothelial cell synthetic microvessel grafts from allogeneic T cells in vivo.

T cell interactions with vascular endothelial cells (EC) are of central importance for immune surveillance of microbes and for pathological processes such as atherosclerosis, allograft rejection, and vasculitis. Animal (especially rodent) models incompletely predict human immune responses, in particular with regard to the immunological functions of EC, and in vitro models may not accurately reflect in vivo findings. In this study, we describe the development of an immunodeficient SCID/bg murine model combining a transplanted human synthetic microvascular bed with adoptive transfer of human T lymphocytes allogeneic to the cells of the graft that more fully recapitulates T cell responses in natural tissues. Using this model, we demonstrate that transduced Bcl-2 protein in the engrafted EC effectively prevents injury even as it enhances T cell graft infiltration and replication.

Animals↗

PubMed2004

AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation.

Previously we have shown that ASK-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP protein family, mediates TNF-induced activation of ASK1-JNK signaling pathway. However, the mechanism by which TNF signaling is coupled to AIP1 is not known. Here we show that AIP1 is localized on the plasma membrane in resting endothelial cells (EC) in a complex with TNFR1. TNF binding induces release of AIP1 from TNFR1, resulting in cytoplasmic translocation and concomitant formation of an intracellular signaling complex comprised of TRADD, RIP1, TRAF2, and AIPl. A proline-rich region (amino acids 796-807) is critical for maintaining AIP1 in a closed form, which associates with a region of TNFR1 distinct from the death domain, the site of TNFR1 association with TRADD. An AIP1 mutant with deletion of this proline-rich region constitutively binds to TRAF2 and ASK1. A PERIOD-like domain (amino acids 591-719) of AIP1 binds to the intact RING finger of TRAF2, and specifically enhances TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK-NF-kappaB signaling. Taken together, our data suggest that AIP1 is a novel transducer in TNF-induced TRAF2-dependent activation of ASK1 that mediates a balance between JNK versus NF-kappaB signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Interferon alpha but not interleukin 12 activates STAT4 signaling in human vascular endothelial cells.

STAT4 signaling, activated by either interleukin 12 (IL12) or interferon alpha (IFNalpha), promotes T(H)1 responses in CD4(+) T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of T(H)1 or T(H)2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNalpha but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rbeta1 and IL12Rbeta2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNalpha responses in STAT4-overexpressing EC; and analysis of IFNalpha responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNalpha but not IL12, and that it may modulate the pro-inflammatory behavior of EC.

Cell Line, Tumor↗

PubMed2004

Endothelial cells present antigens in vivo.

BACKGROUND: Immune recognition of vascular endothelial cells (EC) has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli beta-galactosidase (beta-gal) in two lines of transgenic mice that express beta-gal exclusively in their EC. TIE2-lacZ mice express beta-gal in all EC and VWF-lacZ mice express beta-gal in heart and brain microvascular EC. RESULTS: Transgenic and congenic wild type FVB mice immunized with beta-gal expression vector DNA or beta-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express beta-gal, and their blood vessels showed no histological abnormalities. In response to beta-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-gamma. Infection with recombinant vaccinia virus encoding beta-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express beta-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost beta-gal+ EC and the hosts developed beta-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained beta-gal+ EC and no antisera developed, suggesting a tolerant host immune system. CONCLUSION: Resting, beta-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against beta-gal expressed by EC within transplanted skin. We conclude that EC effectively present intracellular "self" proteins to the immune system. However, antigen presentation by EC does not delete or anergize a large population of specific lymphocytes that respond to the same protein following conventional immunization with protein or expression vector DNA. These results clearly demonstrate striking context sensitivity in the immune recognition of EC, a subtlety that must be better understood in order to treat immune diseases and complications involving the vasculature.

Animals↗

PubMed2004

IL-11 protects human microvascular endothelium from alloinjury in vivo by induction of survivin expression.

IL-11 can reduce tissue injury in animal models of inflammation but the mechanism(s) is unknown. When C.B-17 SCID/beige mice bearing human skin grafts are injected i.p. with human PBMC allogeneic to the donor skin, infiltrating T cells destroy human microvessels by day 21. Intradermal injection of human IL-11 (500 ng/day) delays the time course of graft microvessel loss without reducing the extent of T cell infiltration. Protective actions of IL-11 are most pronounced on day 15. IL-11 has no effect on T cell activation marker, effector molecule, cytokine expression, or endothelial ICAM-1 expression. IL-11 up-regulates the expression of survivin, a cytoprotective protein, in graft keratinocytes and endothelial cells. Topical application of survivin antisense oligonucleotide down-regulates survivin expression in both cell types and largely abrogates the protective effect of IL-11. We conclude that in this human transplant model, IL-11 exerts a cytoprotective rather than anti-inflammatory or immunomodulatory effect mediated through induction of survivin.

Adjuvants, Immunologic↗

PubMed2004

Interferon-gamma plays a nonredundant role in mediating T cell-dependent outward vascular remodeling of allogeneic human coronary arteries.

Vascular remodeling (change in vessel diameter) rather than intimal hyperplasia is the most important predictor of luminal loss in immune-mediated arterial injury, yet little is known about its mechanisms. Here, we show that outward vascular remodeling and intimal thickening, two manifestations of arteriosclerosis with opposing effects on luminal size, result from immune effector mechanisms that are T-cell dependent and interferon (IFN)-gamma mediated. In our in vivo model of human coronary artery injury by allogeneic peripheral blood mononuclear cells, both processes occur concurrently and are characterized by T-cell infiltrates with a predominantly IFN-gamma-producing cytokine profile. Neutralization of IFN-gamma inhibits the arterial and intimal expansion, whereas administration of IFN-gamma enhances these effects. The nonredundant role of IFN-gamma in T-cell-dependent remodeling of human coronary arteries demonstrated here presents a new therapeutic target for preservation of vessel lumen in arteriosclerosis.

Adoptive Transfer↗

PubMed2004

Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-gamma via CXCR3 chemokines.

We investigated the role of T helper (Th)1- and Th2-type cytokines in delayed-type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freund's adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen-specific eosinophil-rich responses were elicited in wild-type mice, but not in T-cell receptor-alpha-/- mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma). IFN-gamma-dependent specific immunoglobulin G (IgG)2a and IL-4-dependent IgG1 were also generated. Delayed-type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL-5-/-, IL-4-/- and signal transducer and activator of transcription-6 (STAT-6)-/- mice, and with anti-IL-4 treatment of wild-type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN-gamma-/- mice, and IFN-gamma protein and the IFN-gamma-inducible CXC chemokine, IP-10, were present in 24-hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP-10. These results suggest that both a Th2-like (IL-5, IL-4 and STAT-6) and a Th1-like (IFN-gamma, IP-10, CXCR3) pathway contribute to eosinophil recruitment in early delayed-type hypersensitivity.

Animals↗

PubMed2004

Airway hyper-reactivity mediated by B-1 cell immunoglobulin M antibody generating complement C5a at 1 day post-immunization in a murine hapten model of non-atopic asthma.

Contact skin immunization of mice with reactive hapten antigen and subsequent airway challenge with the same hapten induces immediate airflow obstruction and subsequent airway hyper-reactivity (AHR) to methacholine challenge, which is dependent on B cells but not on T cells. This responsiveness to airway challenge with antigen is elicited as early as 1 day postimmunization and can be adoptively transferred to naïve recipients via 1-day immune cells. Responses are absent in 1-day immune B-cell-deficient JH(-/-) mice and B-1 B-cell-deficient xid male mice, as well as in recipients of 1-day immune cells depleted of cells with the B-1 cell phenotype (CD19(+) B220(+) CD5(+)). As B-1 cells produce immunoglobulin M (IgM), we sought and found significantly increased numbers of anti-hapten IgM-producing cells in the spleen and lymph nodes of 1-day immune wild-type mice, but not in xid mice. Then, we passively immunized naive mice with anti-hapten IgM monoclonal antibody and, following airway hapten challenge of the recipients, we showed both immediate airflow obstruction and AHR. In addition, AHR was absent in complement C5 and C5a receptor-deficient mice. In summary, this study of the very early elicited phase of a hapten asthma model suggests, for the first time, a role of B-1 cells in producing IgM to activate complement to rapidly mediate asthma airway reactivity only 1 day after immunization.

Animals↗

PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗

PubMed2004

T lymphocyte-endothelial cell interactions.

Human vascular endothelial cells (EC) basally display class I and II MHC-peptide complexes on their surface and come in regular contact with circulating T cells. We propose that EC present microbial antigens to memory T cells as a mechanism of immune surveillance. Activated T cells, in turn, provide both soluble and contact-dependent signals to modulate normal EC functions, including formation and remodeling of blood vessels, regulation of blood flow, regulation of blood fluidity, maintenance of permselectivity, recruitment of inflammatory leukocytes, and antigen presentation leading to activation of T cells. T cell interactions with vascular EC are thus bidirectional and link the immune and circulatory systems.

Animals↗

PubMed2004

T cell-mediated vascular dysfunction of human allografts results from IFN-gamma dysregulation of NO synthase.

Allograft vascular dysfunction predisposes to arteriosclerosis and graft loss. We examined how dysfunction develops in transplanted human arteries in response to circulating allogeneic T cells in vivo using immunodeficient murine hosts. Within 7-9 days, transplanted arteries developed endothelial cell (EC) dysfunction but remained sensitive to exogenous NO. By 2 weeks, the grafts developed impaired contractility and desensitization to NO, both signs of VSMC dysfunction. These T cell-dependent changes correlated with loss of eNOS and expression of iNOS--the latter predominantly within infiltrating T cells. Neutralizing IFN-gamma completely prevented both vascular dysfunction and changes in NOS expression; neutralizing TNF reduced IFN-gamma production and partially prevented dysfunction. Inhibiting iNOS partially preserved responses to NO at 2 weeks and reduced graft intimal expansion after 4 weeks in vivo. In vitro, memory CD4+ T cells acted on allogeneic cultured ECs to reduce eNOS activity and expression of protein and mRNA. These effects required T cell activation by class II MHC antigens and costimulators (principally lymphocyte function-associated antigen-3, or LFA-3) on the ECs and were mediated by production of soluble mediators including IFN-gamma and TNF. We conclude that IFN-gamma is a central mediator of vascular dysfunction and, through dysregulation of NOS expression, links early dysfunction with late arteriosclerosis.

Animals↗

PubMed2003

Endothelial cells require STAT3 for protection against endotoxin-induced inflammation.

Endothelial cells (ECs) are believed to be an important component in the protection from lipopolysaccharide (LPS)-induced endotoxic shock. However, the cellular and molecular mechanism is not well defined. Here, we report that signal transducer and activator of transcription (STAT) 3 is an essential regulator of the antiinflammatory function of ECs in systemic immunity. Because STAT3 deficiency results in early embryonic lethality, we have generated mice with a conditional STAT3 deletion in endothelium (STAT3E-/-). STAT3E-/- mice are healthy and fertile, and isolated ECs initiate normal tube formation in vitro. Conditional endothelial but not organ-specific (i.e., hepatocyte or cardiomyocyte) STAT3 knockout mice show an increased susceptibility to lethality after LPS challenge. The LPS response in STAT3E-/- mice shows exaggerated inflammation and leukocyte infiltration in multiple organs combined with elevated activity of serum alanine aminotransferase and aspartate aminotransferase, indicating organ damage. Concomitantly, proinflammatory cytokines are produced at an exaggerated level and for a prolonged period. This defect cannot be explained by lack of antiinflammatory cytokines, such as interleukin 10 and transforming growth factor beta. Instead, we have shown that a soluble activity derived from endothelia and dependent on STAT3 is critical for suppression of interferon gamma. These data define STAT3 signaling within endothelia as a critical antiinflammatory mediator and provide new insight to the protective function of ECs in inflammation.

Animals↗

PubMed2003

TRAIL induces apoptosis and inflammatory gene expression in human endothelial cells.

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes approximately 30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4',6'-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-kappaB, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL.

Animals↗

PubMed2003

Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H2O2-induced cell death.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with 100% oxygen. Previous studies have determined that both interleukin (IL)-6 and IL-11 are protective in oxygen toxicity. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals, and both IL-6 and IL-11 markedly diminished this cell death response. However, the mechanism(s) by which these cytokines protect cells from death is unclear. In the present study, we characterized the effects of H2O2 on subconfluent human umbilical vein endothelial cell (HUVEC) and human pulmonary microvascular endothelial cell (HPMEC) cultures. We found that preincubation of HUVEC cultures with either IL-6 or IL-11 diminished H2O2 (1.0 mM)-induced cell death. Similar effects were noted with HPMEC showing that this effect is not HUVEC-specific. The protective effects of both IL-6 and IL-11 were not associated with any changes in antioxidants and were decreased by approximately 80% in the presence of U0126, a specific inhibitor of MEK-1-dependent pathways. The cytoprotective effects of IL-11 and IL-6 were also completely eliminated in STAT3 dominant-negative transduced HUVEC cultures. These studies demonstrate that IL-6 and IL-11 both confer cytoprotective effects that diminish oxidant-mediated endothelial cell injury. They also demonstrate that this protection is mediated, at least in part, by a STAT3 and MEK-1-dependent specific signal transduction pathway(s).

Antioxidants↗

PubMed2003

Desensitization of signaling by oncostatin M in human vascular cells involves cytoplasmic Tyr residue 759 in gp130 but is not mediated by either Src homology 2 domain-containing tyrosine phosphatase 2 or suppressor of cytokine signaling 3.

Oncostatin M (OnM) signals through cell surface receptors, which utilize the gp130 subunit. In cultured human umbilical vein endothelial cells (HUVEC), OnM transiently elevates mRNA encoding for suppressor of cytokine signaling-3 (SOCS-3). By 1 h of OnM treatment, HUVEC become refractory to the restimulation by OnM, measured as failure to reinduce SOCS-3 mRNA. OnM-induced desensitization also prevents responses to other gp130-signaling cytokines (e.g. leukemia inhibitory factor and interleukin 11). OnM treatment does not affect gp130 expression levels and desensitizes signaling mediated by a transduced chimeric receptor containing extracellular domains of platelet-derived growth factor receptor-beta (PDGFRbeta) and the cytoplasmic region of gp130. Interestingly, a chimeric PDGFRbeta-gp130 mutant receptor, in which intracellular Tyr residue 759 of gp130 is replaced by a Phe residue, mediates prolonged signaling and is not cross-desensitized by OnM. Phospho-Tyr759 is the binding site for both SOCS-3 and for Src homology domain 2-containing tyrosine phosphatase 2 (SHP-2). In human aortic smooth muscle cells, neither prevention of SOCS-3 protein induction, using STAT3 or SOCS-3 antisense, nor prevention of SHP-2 expression, also with antisense, ablates desensitization. These data suggest that desensitization of vascular cells to OnM is mediated in trans and involves Tyr residue 759 in gp130 but is not mediated by either SOCS-3 or SHP-2, the only two proteins currently known to bind to gp130 at this site.

Animals↗