Ken Wasserman

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.

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)

Tumor-induced immune dysfunction.

Abstract Immune system-based approaches for the treatment of malignant disease over the past decades have often focused on cytolytic effector cells such as cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL and NK cell activity. However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from CTL- or NK cell-based antitumor approaches. The underlying causes of tumor-associated immune suppression of CTL and NK cell activity are discussed, and features of interest shared with HIV infection, leprosy, and rheumatoid arthritis are also be mentioned. Remarkable and very recent observations have shed more light upon the causes of dysfunctional alterations in CTL and NK cells often associated with these diseases, that in turn have suggested new immunotherapeutic approaches for cancer and infectious disease.

Immunosuppression in human tumor-host interaction: role of cytokines and alterations in signal-transducing molecules

Cancer is a disease in which flank systemic immunosuppression, which typically occurs only in the advavced stages, may cause considerable problems, manifested by recurrent infections and possibly also progression of malignant disease. The possibility of finding subtle alterations in the immune system of cancer patients before these are clinically manifest is increasing as more refined and quantitative methods of measuring immune functions are developed. These methods are shedding light upon mechanisms underlying escape from immune surveillance of tumors. The mystery behind the frequent observation of tumor growth in the face of a seemingly normal systemic immune response is slowly being elucidated. Recently there has been a shift in the ;ocus of research interest from systemic to local events during tumor establishment and subsequent growth. Tumor-infiltrating lymphocytes (TIL) are consistently devoid of cellular effector functions unless cultured in vitro in the presence of non-physiological concentrations of cytokines such as interleukin-2 (IL-2) [64]. The low activity of freshly isolated TIL has been ascribed to suppressor lymphocytes or macrophages [22, 23, 42] or to the secretion of suppressor factors by tumor cells [16, 65, 77]. We will not review in detail immunodepression in malignancy [9, 85], but will rather focus on recent research into the mechanisms responsible for decreased immune responses in cancer patients. We submit that suppressed anti-tumor responses can arise from the normal regulation of T and natural killer (NK) cells as mediated by cells, cytokines and other regulatory molecules. In the first part of this review, we will discuss alterations in cytokine production by patient lymphocytes and cytokine expression within tumors, but are well aware of the plethora of other immunosuppressire factors which have been described in human cancer [11, 86]. The second major focus of this review will De on recently described alterations in signal transducing molecules in patient T and NK cells.

RANTES and MCP-1 chemokine plasma levels in chronic renal transplant dysfunction and chronic renal failure

OBJECTIVES Procedures to diagnose renal allograft rejection depend on detection of graft dysfunction due to the presence of mononuclear leukocytic infiltrates. DESIGN AND METHODS In our study, we pursued an immunodiagnostic approach utilizing an ELISA method on plasma samples to monitor patients waiting to undergo transplantation in order to evidence prognostic developments in renal transplantation and, at least, to diagnose renal chronic transplant dysfunction. We analyzed blood levels of two chemokines, RANTES and MCP-1, which are normally overexpressed locally in renal chronic rejection. RESULTS Our results showed that patients affected by chronic renal failure (and waiting for kidney transplant), as well as kidney-grafted patients affected by chronic transplant dysfunction, had plasma levels of RANTES significantly higher than those of controls (patients without acute or chronic pathologies). CONCLUSIONS Our data suggest a simple method to evaluate the plasmatic presence of RANTES, which could be involved in longterm kidney graft failure.

Protection by L-2-oxothiazolidine-4-carboxylic acid of hydrogen peroxide-induced CD3ζ and CD16ζ chain down-regulation in human peripheral blood lymphocytes and lymphokine-activated killer cells

We investigated whether L-2-oxothiazolidine-4-carboxylic acid (OTC) [in the form of Procysteine, kindly donated by Transcend Therapeutics] could protect peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells from CD3zeta and CD16zeta chain down-regulation induced by H2O2 produced by lipopolysaccharide (LPS)-activated autologous monocytes. OTC is known to enhance glutathione production in cells in which glutathione was depleted by reactive oxygen species. Our data showed that OTC induced a significant increase in CD3zeta and CD16zeta chain expression in peripheral blood lymphocytes and LAK cells, respectively, pretreated for 12 hr at 37 degrees. Moreover, OTC significantly protected peripheral blood lymphocytes and LAK against decreased zeta chain expression induced by lipopolysaccharide-activated monocytes or the addition of H2O2 to the culture medium. Our experiments thus suggested that alterations in signal-transducing molecules, such as decreased CD3zeta and CD16zeta expression observed in cytotoxic T lymphocytes and LAK cells in response to oxidative stress, could be prevented by the use of OTC.

Marked Enhancement of the Antigen-Specific Immune Response by Combining Plasmid DNA-Based Immunization with a Schiff Base-Forming Drug

ABSTRACT Although plasmid DNA (pDNA)-based immunization has proven efficacy, the level of immune responses that is achieved by this route of vaccination is often lower than that induced by traditional vaccines, especially for primates and humans. We report here a simple and potent method to enhance pDNA-based vaccination by using two different plasmids encoding viral or bacterial antigens. This method is based on coadministration of low concentrations of a recently described immunopotentiating, Schiff base-forming drug called tucaresol which has led to significant augmentation of antigen-specific humoral and cellular immune responses. Our data suggest that enhancement of the immune response with tucaresol might provide a powerful tool for the further development of pDNA-based immunization for humans.

Conversion of in vitro cultured human monocytes into effective presenters of an HER2/neu-Encoded CTL peptide epitope

Tumour‐derived peptides have been surveyed, in a variety of systems, for their ability to elicit cytokine release from class I restricted T cells. Analogous studies on ovarian carcinoma have employed the antigen‐processing defective T2 cell line. Purified dendritic cells (DC) have been reported to act as highly effective APC. A facile method was developed whereby DC‐like cells were generated from monocyte precursors. Herein, evidence is presented suggesting DC‐like cells are superior to T2 with respect to their ability to present a defined CTL epitope associated with ovarian carcinoma.

Generation and function of bone marrow-derived dendritic cells from CD4/CD8−/− double-knockout mice

We present a novel, simple and straightforward method to obtain mouse bone marrow-derived dendritic cells (DC) from C57Bl/6 CD4/CD8(-/-) double knock-out mice. This new method, involving culture of bone marrow cells in medium supplemented with Interleukin 4 and Granulocyte-Macrophage Colony-Stimulating Factor, does not involve negative immunodepletion of CD4+ and CD8+ populations, or extensive prior manipulations of the starting population. The resulting, loosely adherent cell population, exhibited the morphological characteristics and typical surface markers of DCs, and were endowed with the functional activities characteristic of bone marrow-derived DCs of wild-type mice. Interestingly, LCMV GP33-41 peptide-loaded CD4/CD8(-/-) DCs were efficiently lysed by peptide-specific activated CTLs in vitro. Furthermore, these peptide-loaded CD4/CD8(-/-) DCs induced a peptide-specific CTL response upon immunization of wild-type C57Bl/6 mice.