Islet allografts are subject to rapid rejection through host cellular immune responses involving mononuclear cell recruitment and tissue injury. Interruption of leukocyte recruitment through ...chemokine receptor targeting is of therapeutic benefit in various experimental models, but little is known about the contribution of chemokine pathways to islet allograft rejection. We found that murine islets produce monocyte chemoattractant protein-1 (MCP-1; CCL2) in vitro and that islet allograft rejection was associated with intragraft expression of MCP-1 and its receptor, CCR2. We therefore investigated whether MCP-1 and CCR2 are required for the rejection of fully MHC-disparate islet allografts. Wild-type mice treated with blocking anti-MCP-1 mAb plus a brief, subtherapeutic course of rapamycin had long-term islet allograft survival, in contrast to the effect of treatment with either mAb or rapamycin alone. CCR2(-/-) mice treated with rapamycin also maintained islet allografts long-term. Both MCP/CCR2- and rapamycin-sensitive signals were required for maximal proliferation of alloreactive T cells, suggesting that MCP-1/CCR2 induce rejection by promoting alloreactive T cell clonal expansion and homing and migration. Prolonged islet allograft survival achieved by blockade of the MCP-1/CCR2 pathway plus rapamycin therapy was accompanied by a mononuclear cell infiltrate expressing the inhibitory receptor, programmed death-1 (PD-1), and its ligand (PD-L1, B7-H1), and prolongation of islet allograft survival was abrogated by anti-PD-L1 mAb therapy. These data show that the blockade of MCP-1 binding to CCR2 in conjunction with subtherapeutic immunosuppression can have profound effects on islet allograft survival and implicate the expression of the PD-1/PD-L1 pathway in the regulation of physiologic responses in vivo.
Purpose: Flavopiridol is a potent cyclin-dependent kinase inhibitor with preclinical activity against non-small cell lung cancer (NSCLC),
inhibiting tumor growth in vitro and in vivo by cytostatic ...and cytotoxic mechanisms. A Phase II trial was conducted to determine the activity and toxicity of flavopiridol
in untreated patients with metastatic NSCLC.
Experimental Design: A total of 20 patients were treated with a 72-h continuous infusion of flavopiridol every 14 days at a dose of 50 mg/m 2 /day and a concentration of 0.1–0.2 mg/ml. Dose escalation to 60 mg/m 2 /day was permitted if no significant toxicity occurred. Response was initially assessed after every two infusions; patients
treated longer than 8 weeks were then assessed after every four infusions. Plasma levels of flavopiridol were measured daily
during the first two infusions to determine steady-state concentrations.
Results: This study was designed to evaluate a total of 45 patients in two stages. However, because no objective responses were seen
in the first 20 patients, the early-stopping rule was invoked, and patient accrual was halted. In four patients who received
eight infusions, progression was documented at 15, 20, 40, and 65 weeks, respectively. The most common toxicities included
grade 1 or 2 diarrhea in 11 patients, asthenia in 10 patients, and venous thromboses in 7 patients. The mean ± SD steady-state
concentration of drug during the first infusion was 200 ± 89.9 n m , sufficient for cytostatic effects in in vitro models.
Conclusions: At the current doses and schedule, flavopiridol does not have cytotoxic activity in NSCLC, although protracted periods of
disease stability were observed with an acceptable degree of toxicity.
Recruitment of blood monocytes into the arterial subendothelium is one of the earliest steps in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, is one likely signal ...involved in this process. To test MCP-1’s role in atherogenesis, low density lipoprotein (LDL) receptor–deficient mice were made genetically deficient for MCP-1 and fed a high cholesterol diet. Despite having the same amount of total and fractionated serum cholesterol as LDL receptor–deficient mice with wild-type MCP-1 alleles, LDL receptor/MCP-1-deficient mice had 83% less lipid deposition throughout their aortas. Consistent with MCP-1’s monocyte chemoattractant properties, compound-deficient mice also had fewer macrophages in their aortic walls. Thus, MCP-1 plays a unique and crucial role in the initiation of atherosclerosis and may provide a new therapeutic target in this disorder.
Transcriptional silencing of tumor suppressor genes in association with DNA methylation contributes to malignant transformation. However, the specific DNA methyltransferases that initiate this ...process are unknown. Here we show that a de novo DNA methyltransferase, DNMT3b, substantially contributes to the oncogenic phenotype in a lung cancer model. Normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and activated Ras were immortal, formed colonies in soft agar, and expressed DNMT3b. Antisense suppression of DNMT3b prevented soft agar growth. Furthermore, mouse embryo fibroblasts expressing T antigen and Ras formed soft agar colonies and large tumors, but fibroblasts from Dnmt3b(-/-) mice did not grow in soft agar and were much less tumorigenic in vivo. The tumor suppressor genes, FHIT, TSLC1, and RASSF1A were downregulated in transformed NHBE cells, and antisense DNMT3b treatment resulted in re-expression of FHIT and TSLC1. While expression of TSCL1 correlated with methylation of CpG dinucleotides in its promoter region, the expression of FHIT did not, suggesting that DNMT3b may silence genes by several mechanisms including direct DNA methylation or recruitment of proteins that modify chromatin. Regardless of mechanism, our data indicate that DNMT3b plays an important role in transformation.
Monocyte chemoattractant protein‐1 (MCP‐1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G‐coupled ...receptor, is the only known receptor that functions at physiologic concentrations of MCP‐1. Despite the importance of CCR2 in mediating MCP‐1 responses, several recent studies have suggested that there may be another functional MCP‐1 receptor. Using arterial smooth muscle cells (SMC) from CCR2−/− mice, we demonstrate that MCP‐1 induces tissue‐factor activity at physiologic concentrations. The induction of tissue factor by MCP‐1 is blocked by pertussis toxin and 1,2‐bis(O‐aminophenyl‐ethane‐ethan)‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is Gαi‐coupled and dependent on mobilization of intracellular Ca2+. MCP‐1 induces a time‐ and concentration‐dependent phosphorylation of the mitogen‐activated protein kinases p42/44. The induction of tissue factor activity by MCP‐1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP‐1 receptor that signals at concentrations of MCP‐1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP‐1 in atherosclerotic arteries and in other inflammatory processes.
Chemokines and their receptors play a key role in immune homeostasis regulating leukocyte migration, differentiation, and function. Viruses have acquired and optimized molecules that interact with ...the chemokine system. These virus-encoded molecules promote cell entry, facilitate dissemination of infected cells, and enable the virus to evade the immune response. One such molecule in the murine gammaherpesvirus 68 genome is the M3 gene, which encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and blocks chemokine signaling in vitro. To test the hypothesis that M3 directly interferes with diverse chemokines in vivo, we examined the interaction of M3 with CCL2 and CXCL13 expressed in the pancreas of transgenic mice. CCL2 expression in the pancreas promoted recruitment of monocytes and dendritic cells; CXCL13 promoted recruitment of B and T lymphocytes. Coexpression of M3 in the pancreas blocked cellular recruitment induced by both CCL2 and CXCL13. These results define M3 as multichemokine blocker and demonstrate its use as a powerful tool to analyze chemokine biology.
Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities ...and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.
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