Rosalba Salcedo

Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Induce Expression of CXCR4 on Human Endothelial Cells : In Vivo Neovascularization Induced by Stromal-Derived Factor-1α

The contribution of chemokines toward angiogenesis is currently a focus of intensive investigation. Certain members of the CXC chemokine family can induce bovine capillary endothelial cell migration in vitro and corneal angiogenesis in vivo, and apparently act via binding to their receptors CXCR1 and CXCR2. We used an RNAse protection assay that permitted the simultaneous detection of mRNA for various CXC chemokine receptors in resting human umbilical vein endothelial cells (HUVECs) and detected low levels of only CXCR4 mRNA. Stimulation of HUVECs with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) up-regulated levels of only CXCR4 mRNA. CXCR4 specifically binds the chemokine stromal-derived factor-1α (SDF-1α). Competitive binding studies using 125I-labeled SDF-1α with Scatchard analysis indicated that VEGF or bFGF induced an average number of approximately 16,600 CXCR4 molecules per endothelial cell, with a Kd = 1.23 × 10−9 mol/L. These receptors were functional as HUVECs and human aorta endothelial cells (HAECs) migrated toward SDF-1α. Although SDF-1α-induced chemotaxis was inhibited by the addition of a neutralizing monoclonal CXCR4 antibody, endothelial chemotaxis toward VEGF was not altered; therefore, the angiogenic effect of VEGF is independent of SDF-1α. Furthermore, subcutaneous SDF-1α injections into mice induced formation of local small blood vessels that was accompanied by leukocytic infiltrates. To test whether these effects were dependent on circulating leukocytes, we successfully obtained SDF-1α-induced neovascularization from cross sections of leukocyte-free rat aorta. Taken together, our data indicate that SDF-1α acts as a potent chemoattractant for endothelial cells of different origins bearing CXCR4 and is a participant in angiogenesis that is regulated at the receptor level by VEGF and bFGF.

MyD88-mediated signaling prevents development of adenocarcinomas of the colon: role of interleukin 18

Signaling through the adaptor protein myeloid differentiation factor 88 (MyD88) promotes carcinogenesis in several cancer models. In contrast, MyD88 signaling has a protective role in the development of azoxymethane (AOM)/dextran sodium sulfate (DSS) colitis-associated cancer (CAC). The inability of Myd88−/− mice to heal ulcers generated upon injury creates an altered inflammatory environment that induces early alterations in expression of genes encoding proinflammatory factors, as well as pathways regulating cell proliferation, apoptosis, and DNA repair, resulting in a dramatic increase in adenoma formation and progression to infiltrating adenocarcinomas with frequent clonal mutations in the β-catenin gene. Others have reported that toll-like receptor (Tlr) 4–deficient mice have a similar susceptibility to colitis to Myd88-deficient mice but, unlike the latter, are resistant to CAC. We have observed that mice deficient for Tlr2 or Il1r do not show a differential susceptibility to colitis or CAC. However, upon AOM/DSS treatment Il18−/− and Il18r1−/− mice were more susceptible to colitis and polyp formation than wild-type mice, suggesting that the phenotype of Myd88−/− mice is, in part, a result of their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that impact tissue homeostasis and carcinogenesis.

Role of Chemokines in Angiogenesis: CXCL12/SDF-1 and CXCR4 Interaction, a Key Regulator of Endothelial Cell Responses

Chemokines are small proteins that act as cell attractants via the activation of G protein‐coupled receptors. Chemokines play an important role in several pathophysiological processes such as inflammation and immunity. Many proinflammatory chemokines also support the development of vascular blood supply at the site of inflammation. Similarly, tumor‐generated chemokines can contribute to tumor growth by promoting angiogenesis. Recently, significant advances have been made in understanding the contribution of chemokines to the angiogenesis process. This review will discuss first the evidence supporting the direct contribution of different chemokine subfamily members, including CC, CXC, and CX3C chemokines, as positive or negative regulators of the angiogenesis process based on the expression of their cognate receptors on endothelial cells. Additionally, the relationship between classic angiogenic factors and chemokine receptor expression on endothelial cells, and the implications of chemokine production by cancer cells will be analyzed with particular emphasis on the CXCL12/stromal‐cell derived factor‐1 interaction with CXCR4.

Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells

The basis for the angiogenic effects of CXC chemokines such as interleukin 8 (IL‐8) and for angiostatic chemokines such as interferon‐inducible protein 10 (IP‐10) has been difficult to assess. We recently reported, based on an RNase protection assay, that human umbilical vein endothelial cells (HUVECs) did not express detectable mRNA for the IL‐8 receptors CXCR1 and CXCR2. This raised the possibility of heterogeneity of receptor expression by different endothelial cell (ECs) types. Since systemic angiogenesis induced by IL‐8 would more likely involve microvessel ECs, we investigated CXC receptor expression on human microvascular dermal endothelial cells (HMECs). By confocal microscopy and immunofluorescence we observed that HMECs consistently expressed high levels of CXCR1 and CXCR4 (mean fluorescence intensity of 261 ±22.1 and 306.2±19, respectively) and intermediate levels of CXCR3 and CXCR2 (173.9±30.2 and 156±30.9, respectively). In contrast, only a small proportion of HUVEC preparations expressed low levels of CXCR1, ‐2, and ‐3 (66±19.9; 49±15, and 81.4±17.9, respectively). However, both HMECs and HUVECs expressed equal levels of CXCR4. As expected, HMECs had more potent chemotactic responses to IL‐8 than HUVECs, and this was correlated with the levels of IL‐8 receptors on the ECs. Antibodies to CXCR1 and CXCR2 each had inhibitory effects on chemotaxis of HMECs to IL‐8, indicating that both IL‐8 receptors contributed to the migratory response of these cells toward IL‐8. Assessment of the functional capacity of CXCR3 unexpectedly revealed that HMECs migrated in response to relatively higher concentrations (100–500 ng/ml) of each of the ‘angiostatic’ chemokines IP‐10, ITAC, and MIG. Despite this, the ‘angiostatic’ chemokines inhibited the chemotactic response of HMECs to IL‐8. IL‐8 and SDF‐1α but not IP‐10 induced calcium mobilization in adherent ECs, suggesting that signaling events associated with calcium mobilization are separable from those required for chemotaxis. Taken together, our data indicated that functional differences among EC types is dependent on the level of the expression of CXC chemokine receptors. Whether this heterogeneityin receptor expression by ECs reflects distinct differentiation pathways remains to be established.—Salcedo, R., Resau, J. H., Halverson, D., Hudson, E. A., Dambach, M., Powell, D., Wasserman, K., Oppenheim, J. J. Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB J. 14, 2055–2064 (2000)

Immunologic and Therapeutic Synergy of IL-27 and IL-2: Enhancement of T Cell Sensitization, Tumor-Specific CTL Reactivity and Complete Regression of Disseminated Neuroblastoma Metastases in the Liver and Bone Marrow

IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8+ ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4+CD25+Foxp3+ regulatory and IL-17-expressing CD4+ cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

MyD88 and its divergent toll in carcinogenesis

Toll-like and interleukin-1 (IL-1) family receptors recognize microbial or endogenous ligands and inflammatory mediators, respectively, and with the exception of Toll-like receptor 3 (TLR3), signal via the adaptor molecule myeloid differentiation factor 88 (MyD88). MyD88 is involved in oncogene-induced cell intrinsic inflammation and in cancer-associated extrinsic inflammation, and as such MyD88 contributes to skin, liver, pancreatic, and colon carcinogenesis, as well as sarcomagenesis. MyD88 is also protective, for example in oncogenic virus carcinogenesis or, acting downstream of IL-18R to strengthen mucosal repair, in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon carcinogenesis. Here, we discuss the mechanisms of the divergent effects of MyD88 and the balance of its protumor role in cancer-enhancing inflammation and immunity and its antitumor role in tissue homeostasis, repair, and immunity against the tumor or oncogenic pathogens.

Increased expression and secretion of ICAM‐1 during experimental infection with Trypanosoma cruzi

In the present study we demonstrate that spleens and hearts from BALB/c mice infected with the virulent Tulahuén or the low virulent CA‐I strains of Trypanosoma cruzi, contain substantially higher ICAM‐1 transcripts than uninfected controls. ICAM‐1 expression in heart cells was also increased at the protein level, as measured by flow cytometry, ELISA and immunohistochemistry. The adhesive receptor was observed not only on inflammatory cells but also on sarcolemma of cardiac myocytes from T. cruzi infected mice. ICAM‐1 expression was higher during the acute phase than in the chronic phase of infection, and paralleled the density of inflammatory leukocytes. Elevated titres of soluble ICAM‐1 (s‐ICAM‐1) were detected in sera from mice during the acute phase of infection with CA‐I or Tulahuén parasites. Cytokines, including IFN‐γ, IL‐1α, IL‐6 and TNF‐α have been shown to modulate expression of ICAM‐1. Spleens and hearts from mice infected with CA‐I or Tulahuén strains showed increased accumulation of mRNAs specific for these cytokines, which peaked during the acute phase of infection. However, IFN‐γ activity was not necessary for ICAM‐1 induction, as its levels were also increased during infection in IFN‐γ receptor knock‐out (IFN‐γR−) mice. Upregulation of ICAM‐1 expression might be a direct consequence of parasite infection, since its density on cell lines of different lineages was enhanced after 24 or 48 h of infection with T. cruzi

Constitutive αVβ3 integrin-mediated adhesion of human lymphoid B cells to vitronectin substrate

Abstract Adherence to cells and matrices participates in lymphocyte migration and tissue localization and contributes to the regulation of growth and differentiation of the lymphoid cells. The adherence is mainly mediated by three families of cell-surface proteins: integrins, immunoglobulin (Ig)-related molecules, and selectins. Integrins recognize Ig-related molecules such as ICAMs as well as fibronectin (FN), vitronectin (VN), and other matrix proteins. In this study, the in vitro adhesive properties of two Epstein—Barr virus-carrying B lymphoblastoid cell lines, IB-4 and NAD-20, were compared. IB-4 cells grow as a monolayer in contrast to NAD-20 cells, which grow as cell clusters. IB-4 cells were found to adhere to the tissue culture vessel through a component of the fetal bovine serum. By using blocking monoclonal antibodies to cell-surface molecules and serum proteins, IB-4 cells were found to use αVβ3 integrin (CD51/CD61) and serum VN as the adhesive molecules. αVβ3 integrin also mediated adhesion of IB-4 cells to human serum VN and to purified VN and FN. This constitutive adherence was not enhanced by phorbol ester treatment and was inhibited by RGD-containing peptides, in contrast to the homotypic adhesion of NAD-20 cells, which was mediated by β2 integrin CD11a/CD18 and its ligand ICAM-1 (CD54). Since VN is a component of both lymphoid tissue matrix and plasma, adhesion to this protein may affect functions and activities of B lymphocytes.

Microbes and Cancer

Commensal microorganisms (the microbiota) live on all the surface barriers of our body and are particularly abundant and diverse in the distal gut. The microbiota and its larger host represent a metaorganism in which the cross talk between microbes and host cells is necessary for health, survival, and regulation of physiological functions locally, at the barrier level, and systemically. The ancestral molecular and cellular mechanisms stemming from the earliest interactions between prokaryotes and eukaryotes have evolved to mediate microbe-dependent host physiology and tissue homeostasis, including innate and adaptive resistance to infections and tissue repair. Mostly because of its effects on metabolism, cellular proliferation, inflammation, and immunity, the microbiota regulates cancer at the level of predisposing conditions, initiation, genetic instability, susceptibility to host immune response, progression, comorbidity, and response to therapy. Here, we review the mechanisms underlying the interaction of the microbiota with cancer and the evidence suggesting that the microbiota could be targeted to improve therapy while attenuating adverse reactions.

The pivotal role of the Leu-CAM and ICAM molecules in human leukocyte adhesion

Cellular adhesion is of fundamental importance in leukocyte physiology. It is a complex, strictly regulated process, which involves the participation of several cell surface glycoproteins. Among the most important are the Leu-CAMs or the CD11/CD18 integrin receptors, and their adhesion ligands ICAM-1 (CD54) and ICAM-2. In this review we summarize some recent work on various aspects of these molecules.