Biomedical subjects

Pablo Engel

Publications and source records attributed to Pablo Engel.

At least 19 recordsLinked to original sources

PubMed2007

Identification and functional characterization of the hepatic stellate cell CD38 cell surface molecule.

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

ADP-ribosyl Cyclase↗

PubMed2006

CD molecules 2006--human cell differentiation molecules.

The Human Leucocyte Differentiation Antigens Workshops (HLDA) have since 1984 provided a forum for the characterization and study of leucocyte surface molecules and antibodies against them. HLDA devised the CD nomenclature, which is sanctioned by IUIS. The HLDA Council reviewed and modified the objectives of HLDA in 2004, and changed the name of the organization to Human Cell Differentiation Molecules (HCDM) to reflect the broader objectives. Workshop studies under the HCDM banner proceeded during 2005 and early 2006, culminating in a meeting in May 2006. At that meeting the Council, acting as Nomenclature Committee, approved a number of new CD designations and changes to some pre-existing CD designations, which are summarized in this report.

Antigens, CD↗

PubMed2005

Cutting edge: MyD88 controls phagocyte NADPH oxidase function and killing of gram-negative bacteria.

MyD88 is an adaptor protein for the TLR family of proteins that has been implicated as a critical mediator of innate immune responses to pathogen detection. In this study, we report that MyD88 plays a crucial role in killing Gram-negative bacteria by primary macrophages via influencing NADPH oxidase function. Peritoneal macrophages from MyD88-/- mice exhibited a marked inability to kill Escherichia coli (F18) or an attenuated strain of Salmonella typhimurium (sseB) in vitro. This defect in killing was due to diminished NADPH oxidase-mediated production of superoxide anion in response to bacteria by MyD88-/- phagocytes as a consequence of defective NADPH oxidase assembly. Defective oxidase assembly in MyD88-deficient macrophages resulted from impaired p38 MAPK activation and subsequent phosphorylation of p47phox. Together these data demonstrate a pivotal role for MyD88 in killing Gram-negative bacteria via modulation of NADPH oxidase activity in phagocytic cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing.

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Characterization of antibodies submitted to the B cell section of the 8th Human Leukocyte Differentiation Antigens Workshop by flow cytometry and immunohistochemistry.

The aim of this study was to characterize the reactivity of monoclonal antibodies (mAbs) that had been submitted to the HLDA8 Workshop. The lineage specificity of target molecules was tested by analyzing their expression patterns on blood cells, leukocytes, and lymphocyte subsets. The expression of target molecules during B cell development, ranging from early precursors to plasma cells, was analyzed using a large panel of B cell lines. Our results have permitted us to characterize the expression of 10 new CD molecules: CD316 (HM1.24, BST2), CD268 (BAFF-R, TNFRSF13C), CD269 (BCMA, TNFRF17), CD267 (TACI, TNFRSF13B), CD275 (ICOSL, B7H2), CD254 (TRANCE, TNFSF11), CD252 (OX40L TNFSF4), CD315 (CD9-P), CD316 (EWI-2, PGRL), and CD307 (IRTA-2 or FcRH5). Three of these new CDs, CD267, CD269, and CD307 presented a B cell-restricted expression pattern. MAbs against these novel cell-surface molecules may offer new tools for research, diagnosis, and therapy.

Animals↗

PubMed2005

CD molecules 2005: human cell differentiation molecules.

The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.

Antigens, CD↗

PubMed2005

CD229 (Ly9) lymphocyte cell surface receptor interacts homophilically through its N-terminal domain and relocalizes to the immunological synapse.

CD229 is a member of the CD150 family of the Ig superfamily expressed on T and B cells. Receptors of this family regulate cytokine production and cytotoxicity of lymphocytes and NK cells. The cytoplasmic tail of CD229 binds to SAP, a protein that is defective in X-linked lymphoproliferative syndrome. To identify the CD229 ligand, we generated a soluble Ig fusion protein containing the two N-terminal extracellular domains of human CD229 (CD229-Ig). CD229-Ig bound to CD229-transfected cells, whereas no binding was detected on cells expressing other CD150 family receptors, showing that CD229 binds homophilically. Both human and mouse CD229 interacted with itself. Domain deletion mutants showed that the N-terminal Ig-domain mediates homophilic adhesion. CD229-CD229 binding was severely compromised when the charged amino acids E27 and E29 on the predicted B-C loop and R89 on the F-G loop of the N-terminal domain were mutated to alanine. In contrast, one mutation, R44A, enhanced the homophilic interaction. Confocal microscopy image analysis revealed relocalization of CD229 to the contact area of T and B cells during Ag-dependent immune synapse formation. Thus, CD229 is its own ligand and participates in the immunological synapse.

Animals↗

PubMed2005

Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229.

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.

Adaptor Protein Complex 2↗

PubMed2005

Induction of tumor NK-cell immunity by anti-CD69 antibody therapy.

The leukocyte activation marker CD69 is a novel regulator of the immune response, modulating the production of cytokines including transforming growth factor-beta (TGF-beta). We have generated an antimurine CD69 monoclonal antibody (mAb), CD69.2.2, which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell-dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69(-/-) mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-beta production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor-independent manner, resulting in a substantial increase in both NK-cell cytolytic activity and interferon gamma (IFNgamma) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis.

Animals↗

PubMed2005

Effects of cryopreservation on the immunogenicity of porcine arterial allografts in early stages of transplant vasculopathy.

BACKGROUND: The number of revascularization procedures including coronary and lower extremity bypass, have increased greatly in the last decade. It suggests a growing need for vascular grafts. Cryopreserved allografts could represent a viable alternative but their immunologic reactivity remains controversial. METHODS: 71 pigs (40 recipients and 31 donors) were used. Two femoral grafts per recipient animal were implanted for 3, 7, and 30 days. Types of grafts: fresh autograft as a control graft (n=19), fresh allograft (n=31) and cryopreserved allograft (n=30). Histological and immunohistochemical studies were performed. RESULTS: Fresh allografts compared to autografts showed intimal inflammatory infiltration at 3 days (328 vs. 0 macrophages/mm2; P<0.05) and 7 days (962 vs. 139 T lymphocytes/mm2; P<0.05) post-transplantation. At 30 days, there was a loss of endothelial cells, presence of luminal thrombus and aneurismal lesions (total area=15.8 vs. 8.4 mm2; P<0.05). Cryopreservation did not reduce these lesions nor modify endothelial nitric oxide synthase (eNOS) expression nor modify the number of animals that developed anti-SLA antibodies. Moreover, at 7 days, cryopreserved allografts compared to fresh allografts showed a higher expression of P-selectin (5 out of 5 vs. 1 out of 5; P<0.05) and, at 30 days, a greater inflammatory reactivity (2692 vs. 1107 T lymphocytes/mm2 in media; P<0.05) with a trend towards a higher presence of multinucleated giant cells than in the fresh ones. CONCLUSIONS: The cryopreservation method used maintained immunogenicity of allografts and increased the inflammatory reactivity found in fresh allografts up to 30 days of vascular transplantation.

Aneurysm↗

PubMed2005

P-selectin mediates leukocyte rolling in concanavalin-A-induced hepatitis.

UNLABELLED: Concanavalin-A (Con-A)-induced hepatitis is an experimental model of human autoimmune hepatitis characterized by leukocyte activation and infiltration of the liver. The aim of the present study was to evaluate the role of P-selectin on leukocyte-endothelial interactions within the hepatic microvasculature in response to Con-A. METHODS: The study was performed in P-selectin-deficient mice and wild-type mice pretreated with anti-P-selectin blocking monoclonal antibody (mAb) or vehicle. After 2 h of Con-A (20 mg/kg i.v.) or PBS administration, leukocyte rolling and adhesion and the index of sinusoidal perfusion were evaluated using the intravital microscopy technique in the liver. Apoptosis was determined by flow cytometry analysis of caspase-3 activity assayed on freshly isolated hepatocytes. RESULTS: Con-A induced a significant increase in leukocyte rolling, mainly located at the central venule (2.1+/-0.4 vs 0.6+/-0.2 cells/min in wild-type mice treated with vehicle) and less marked, but still significant, in portal venules. This was associated with a significant increase in leukocyte adhesion. In P-selectin-deficient mice treated with Con-A, leukocyte rolling in portal and central venules was markedly reduced. However, leukocyte adhesion was only partially attenuated. A few sinusoids were perfused in wild-type mice treated with Con-A (26%). The percentage of perfused sinusoids was significantly higher in P-selectin-deficient mice (45%; P<0.05 vs wild-type). Similar effects were noted after the simultaneous injection of Con-A and anti-P-selecting mAb in wild-type mice. After Con-A treatment, apoptosis was markedly reduced in isolated hepatocytes of P-selectin-deficent mice (37+/-7% vs 75+/-5% in wild type). CONCLUSION: The results of this intravital microscopy study clearly demonstrate that P-selectin is involved in the initial leukocyte rolling that leads to the development of Con-A-induced liver injury.

Animals↗

PubMed2004

Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers.

CCl(4) cirrhotic rat liver exhibits a hyperresponse to the alpha(1)-adrenergic agonist methoxamine (Mtx) that is associated with enhanced thromboxane A(2) (TXA(2)) production and is abrogated by indomethacin. To further elucidate the molecular mechanisms involved in the hyperresponse to vasoconstrictors, portal perfusion pressure dose-response curves to Mtx were performed in CCl(4) cirrhotic rats livers after preincubation with vehicle, the cyclooxygenase (COX)-1 selective inhibitor SC-560, and the COX-2 selective inhibitor SC-236. TXA(2) production was determined in samples of the perfusate. COX-1 expression was analyzed and quantified in hepatocytes, Kupffer cells, sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) isolated from control and cirrhotic rat livers by double-immunofluorescence staining, with specific markers for each population using flow cytometry or Western blot analysis. COX-1 protein levels were not significantly increased in cirrhotic livers, but COX-2 protein expression was increased. COX-1 inhibition, but not COX-2, significantly attenuated the response to Mtx and prevented the increased production of TXA(2). Cirrhotic livers showed an increased expression of COX-1 in SEC and reduced expression in HSC compared with control livers, whereas COX-1 was similarly distributed in Kupffer cells. Despite abundant hepatic COX-2 expression, the increased response to Mtx of cirrhotic livers is mainly dependent of COX-1. Upregulation of COX-1 in cirrhotic SEC may be responsible for the hyperesponse to Mtx.

Animals↗

PubMed2004

Identification and characterization of a novel spliced variant that encodes human soluble tumor necrosis factor receptor 2.

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation and cell death induction in several cell types. The biological effects of TNF-alpha are mediated via the cell-surface TNF receptors TNFR1 and TNFR2. Soluble forms of these two receptors, which contain the extracellular ectodomains, are proteolytically cleaved from the membrane. High levels of soluble (s) TNFR2 in serum have been documented in multiple inflammatory pathologies. We describe here a new differential spliced isoform of human TNFR2 missing exons 7 and 8, DS-TNFR2(Delta7,8). This novel isoform lacks the transmembrane and cytoplasmic domains. Expression studies with DS-TNFR2(Delta7,8) cDNA transiently transfected COS cells showed that it encodes a sTNFR2 receptor of approximately 42 kDa. Soluble DS-TNFR2(Delta7,8) blocked TNF-alpha-induced apoptosis, which suggests that it regulates TNF-alpha function by antagonizing its biological activity. An ELISA was developed that quantifies sTNFR2 generated by alternative splicing. Our data show that sTNFR2 generated by alternative splicing can be found in sera of healthy individuals, at increased levels in patients with sepsis and at high concentrations in rheumatoid arthritis patients.

Adult↗

PubMed2003

Characterization of platelet and soluble-porcine P-selectin (CD62P).

P-selectin (CD62P), an adhesion molecule expressed on activated endothelial cells and platelets, mediates the initial attachment of leukocytes to the stimulated endothelium upon inflammation and the interaction between leukocytes and platelets. A soluble form of P-selectin is present in the serum of healthy individuals as a circulating protein and high levels have been described in various pathological situations. The aim of this study was to characterize P-selectin on porcine platelets and investigate the soluble form of this protein, which are uncharacterized in several animal species including pigs. A new monoclonal antibody (mAb) (SwPsel.1.9) against porcine P-selectin was produced using a mouse cell line transfected with pig P-selectin cDNA. This mAb together with a previously described mAb (P-sel.KO.2.5), produced in our laboratory, was used to develop an ELISA to quantify porcine P-selectin. No significant levels of soluble-porcine P-selectin were observed in healthy animals. However, the total amount of P-selectin measured in porcine platelets was similar to that found in humans. Increased levels of this circulating protein were detected in the plasma from pigs after allograft implantation. In vitro, P-selectin expression on platelet membrane was rapidly induced by PMA and thrombin, as assessed by flow cytometry. However, these activators did not stimulate the release of soluble P-selectin. Analysis of the proteolytic cleavage of this protein from COS-transfected cells revealed that PMA treatment failed to cause the shedding of membrane-bound P-selectin. These data suggest that porcine P-selectin is a suitable marker for inflammation and that the mechanism involved in the generation of circulating P-selectin is not proteolytic release.

Animals↗

PubMed2003

Relative roles of ICAM-1 and VCAM-1 in the pathogenesis of experimental radiation-induced intestinal inflammation.

PURPOSE: Cell adhesion molecules mediate leukocyte recruitment into the irradiated organs; modulation of this process may protect from radiation damage. Our objective was to characterize the requirement for intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in intestinal inflammatory response after abdominal irradiation. METHODS AND MATERIALS: Endothelial ICAM-1 and VCAM-1 expression was determined using radiolabeled antibodies in mice 24 h and 14 days after irradiation with 10 Gy, or sham radiation. Leukocyte-endothelial cell interactions in intestinal venules were assessed using intravital microscopy, and the function of ICAM-1 and VCAM-1 in this process by using blocking antibodies and ICAM-1(-/-) mice. RESULTS: The number of adherent leukocytes significantly increased 24 h after irradiation and remained elevated at 14 days. Treatment with anti-ICAM-1 antibodies and ICAM-1 genetic deficiency significantly reduced leukocyte adhesion 24 h after irradiation. At 14 days after irradiation, both wild-type and ICAM-1(-/-) mice had an upregulation of VCAM-1, expression, and VCAM-1 immunoneutralization, but not ICAM-1 immunoneutralization, significantly reduced leukocyte adhesion. In ICAM-1(-/-) mice, regeneration of the intestinal epithelium was enhanced relative to wild-type mice. CONCLUSIONS: ICAM-1 plays a key role in leukocyte recruitment at early time points after abdominal irradiation, whereas VCAM-1 is the main molecular determinant of leukocyte recruitment at late time points.

Animals↗

PubMed2003

Enhanced antitumor immunity in mice deficient in CD69.

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated, and was due, at least in part, to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1, diminished TGF-beta production, and decreased lymphocyte apoptosis. Moreover, the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition, CD69 engagement induced NK and T cell production of TGF-beta, directly linking CD69 signaling to TGF-beta regulation. Furthermore, anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors.

Animals↗

PubMed2003

The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2).

CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.

Adaptor Protein Complex 2↗

PubMed2003

The SAP and SLAM families in immune responses and X-linked lymphoproliferative disease.

SAP (signalling lymphocytic activation molecule (SLAM)-associated protein) is a T- and natural killer (NK)-cell-specific protein containing a single SH2 domain encoded by a gene that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). The SH2 domain of SAP binds with high affinity to the cytoplasmic tail of the haematopoietic cell-surface glycoprotein SLAM and five related receptors. SAP regulates signal transduction of the SLAM-family receptors by recruiting SRC kinases. Similarly, the SAP-related proteins EAT2A and EAT2B are thought to control signal transduction that is initiated by SLAM-related receptors in professional antigen-presenting cells. In this review, we discuss recent findings on the structure and function of proteins of the SAP and SLAM families.

Animals↗