Evidence indicates that gaining weight in adult life is associated with an elevated risk of colorectal cancer; however, biological mechanisms that may explain this association remain unclear. We evaluated the mediation effect of 20 different biomarkers on the relationship between adult weight gain and colorectal cancer, using data from a prospective nested case-control study of 452 incident cases diagnosed between 1992 and 2003 and matched within risk sets to 452 controls within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. The proportions of mediated effects (%) were estimated on the basis of differences in percent effect changes in conditional logistic regression models with and without additional adjustment for individual biomarkers. Greater adult weight gain (≥300 g/year vs. <300 g/year) was associated with a higher risk of colon cancer (multivariable-adjusted relative risk = 1.54, 95% confidence interval: 1.07, 2.24) but not rectal cancer (relative risk = 1.07, 95% confidence interval: 0.68, 1.66). This association was accounted for mostly by attained waist circumference (reduction of 61%) and by the biomarkers soluble leptin receptor (reduction of 43%) and glycated hemoglobin (reduction of 28%). These novel data suggest that the observed association between adult weight gain and colon cancer could be primarily explained by attained abdominal fatness and biomarkers of metabolic dysfunction.

Molecules representative of different classes of chemotactic agents, including formyl-Met-Leu-Phe (FMLP), C5a, leukotriene B4, platelet-activating factor, and interleukin (IL)-8, caused a rapid reduction in the IL-1 binding capacity by human polymorphonuclear leukocytes (PMN), a cell type expressing predominantly the IL-1 type II decoy receptor (IL-1 decoy RII). N-t-Boc-Met-Leu-Phe, an antagonist for the FMLP receptor, inhibited the loss of IL-1 binding capacity induced by FMLP. Monocyte chemotactic protein 1, a chemokine related to IL-8 but inactive on PMN, had no effect on IL-1 binding in this cell type. FMLP was selected for further detailed analysis of chemoattractant-induced loss of IL-1 binding by PMN. The action of FMLP was rapid, reaching 50% of its maximum (80%) at 30 s, the earliest measurable time point, and plateauing between 10 and 30 min. Dose-response analysis revealed that maximal reduction of IL-1 binding was reached at FMLP concentrations that were also optimal for chemotaxis (50% effective dose = 5 x 10(-9) M). The loss of IL-1 binding capacity caused by FMLP was determined by a reduction in receptor number with no change in their affinity. The effect of FMLP on IL-1 receptor (IL-1R) was selective in that the PMN surface structures IL-8R, CD16, CD18, and major histocompatibility complex class I antigens were unaffected under these conditions. Loss of surface IL-1R was not due to an augumented rate of internalization. FMLP caused rapid release of a 45-kD IL-1-binding molecule identified as the IL-1 decoy RII. After FMLP-induced release, PMN reexpressed newly synthesized receptors, reaching basal levels by 4 h. FMLP-induced release of the IL-1 decoy RII did not impair the responsiveness of PMN to IL-1 in terms of promotion of survival and cytokine production. FMLP-induced release of the IL-1 decoy RII was unaffected by protein synthesis inhibitors, was blocked by certain protease inhibitors, and was mimicked by agents (the Ca++ ionophore A23187 and phorbol myristate acetate) that recapitulate elements in the signal transduction pathway of chemoattractant receptors. The time frame and concentration range of chemoattractant-induced rapid release of the IL-1 decoy RII are consistent with the view that IL-1 decoy RII release is an early event in the multistep process of leukocyte recruitment.(ABSTRACT TRUNCATED AT 400 WORDS)

By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.

Endothelial cell-specific molecules are potential targets for new therapeutic strategies in the control of inflammatory reactions, immune responses and neoangiogenesis. We describe the production and characterization of MEC 14.7, a monoclonal antibody directed to murine endothelial cells recognizing a glycosylated protein with an apparent molecular mass of about 100 kDa in cultured endothelioma cell lysate and about 80 kDa in lung lysate. MEC 14.7 antigen was selectively expressed by the endothelium in vivo, particularly in small vessels and neoformed capillaries and by developing vascular structures in embryonal bodies. Deglycosylation of the molecule with neuraminidase, O- and N-glycanase showed that the MEC 14.7 epitope is neuraminidase-sensitive. MEC 14.7 antigen was purified from lung lysates by chromatographic techniques, and sequenced internal peptides indicated it was identical with murine CD34. Thus the apparent molecular mass of CD34 is heterogeneous, depending on the glycosylation state in the different cell types. Immunomagnetic isolation and culture of MEC 14.7-positive bone marrow cells showed that this antibody recognizes hematopoietic progenitors (particularly myelomonocytic) and can be used in murine models of bone marrow reconstitution.

The mannose receptor (MR) is a surface 175-kd C-type lectin expressed by macrophages and dendritic cells. MR is involved in removal of effete cells, phagocytosis of mannose-coated particles, pinocytosis, and antigen presentation. Expression of MR was investigated in 17 biopsies of Kaposi's sarcoma (3 AIDS KS, 13 classical KS, and 1 transplant-associated KS) using three anti-MR monoclonal antibodies (3.29, D547, and PAM1). Immunostaining for MR was detected in 94 +/- 7% KS cells with spindle morphology. In normal tissues, MR was expressed by sinus-lining cells of spleen and lymph nodes, but it was not detected in endothelial cells lining normal hematic and lymphatic vessels, hemangioma, hemangioendothelioma, and lymphangioma. Expression of MR in KS cells prompted us to investigate the possibility that they derive from a circulating precursor cell. Peripheral blood mononuclear cells from 16 patients with KS (10 classical, 1 transplanted, and 5 AIDS) were cultured in PHA-conditioned medium for 10 to 14 days. Confluent monolayers of adherent spindle cells were detected in 8 of 11 classical KS, in 5 of 5 AIDS KS patients, and in 0 of 34 control patients. Peripheral-blood-derived KS-like cells were characterized by co-expression of macrophage and endothelial antigens being positive for CD45 (60%), CD68 (98%), MR (70%), CD14 (25%), VE-cadherin (70%), and von Willebrand factor (10%). When the immunophenotype of peripheral-blood-derived adherent cells was compared with that of KS spindle cells of tissue biopsies, it was found that both cell types are VE-cadherin+/MR+/CD68+, that peripheral-blood-derived spindle cells are CD34- and are less frequently stained for CD31 and von Willebrand factor, and that lesional KS cells do not express the leukocyte markers CD45 and CD18. Our findings are consistent with the possibility that KS lesions derive from tissue accumulation and local proliferation of a special subset of macrophages with endothelial features the normal counterpart of which are the sinus-lining cells of spleen and lymph nodes.

Abstract Human neutrophils (polymorphonuclear leukocytes; PMN) respond to some CXC chemokines but do not migrate to CC chemokines. Recent work has shown that chemokine receptors can be modulated by inflammatory cytokines. In this study, the effect of IFN-γ, a prototypic Th1 cytokine, on chemokine receptor expression in PMN was investigated. IFN-γ caused a rapid (∼1 h) and concentration-dependent increase of CCR1 and CCR3 mRNA. The expression of CCR2, CCR5, and CXCR1–4 was not augmented. IFN-γ-treated PMN, but not control cells, expressed specific binding sites for labeled monocyte-chemotactic protein (MCP)-3 and migrated to macrophage-inflammatory protein (MIP)-1α, RANTES, MCP-3, MIP-5/HCC2, and eotaxin. 7B11, a mAb for CCR3, inhibited the chemotactic response of IFN-γ-treated PMN to eotaxin, and aminoxypentane-RANTES blocked PMN migration to RANTES. These results suggest that the selectivity of certain chemokines for their target cells may be altered by cytokines produced within an inflammatory context. Since PMN may play a role in orienting immunity toward Th1 responses, it is possible to speculate that IFN-γ not only promotes Th1 differentiation directly, but also reorients the functional significance of Th2 effector cytokines by broadening the spectrum of their action to include PMN.

Under certain conditions, natural killer (NK) cells accumulate rapidly at extrahematic sites. In an effort to define the mechanisms underlying the recruitment of NK cells in tissues, we investigated their ability to adhere and transmigrate across endothelial cell (EC) monolayers. A considerable proportion of NK cells adhered to EC and about 30-40% of the adherent NK cells transmigrated across EC. NK cells were 2-3 times more efficient than resting T cells. Exposure of NK cells to IL-2 and IL-12 augmented their adhesive ability, while IL-4 had an inhibitory effect. mAb directed against CD18 and CD11a inhibited binding and migration of NK cells across resting or IL-1-activated EC, whereas anti-CD11b and Cd11c did not. Using IL-1-activated EC, it was found that anti-VLA-4 and anti-VCAM-1 mAb utilized in concert with anti-CD18 significantly reduced adhesion and transmigration. The CS-1 peptide of fibronectin (which recognizes VLA-4), when used in concert with anti-CD18 and anti-VCAM-1 (but not anti-VLA-4), caused a small, but significant increase in inhibition. Thus, LFA-1 and VLA-4 are crucial determinants of the adhesive and migratory interaction with the vascular endothelium.

Besides the regulation of hematopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF)induces the expression of a functional program in endothelial cells (ECs) related to angiogenesis and to their survival in the bone marrow microenvironment. ECs express specific GM-CSF high-affinity binding sites, which mediate the proliferative and migratory response. We now report that ECs express the alpha and beta subunits of GM-CSF receptor (GM-CSFR), and that GM-CSF is able to activate the Janus kinase (JAK)2, a member of the cytosolic tyrosine kinase family, which is known to mediate signals of several non-tyrosine kinase receptors. JAK2 tyrosine phosphorylation, as well as activation of its catalytic activity, is induced by subnanomolar concentrations of GM-CSF and occurs within 3 minutes of stimulation and persists at least for 10 minutes. The effect is specific as inferred by the lack of effect of heat-inactivated GM-CSF or neutralized by specific antibodies and by the finding that interleukin-5, which utilizes a specific alpha chain and the same beta chain of GM-CSFR, does not phosphorylate JAK2. Furthermore, we show that the amount of JAK2 physically associated with GM-CSFR beta chain is increased after GM-CSF stimulation and that GM-CSF triggers both beta chain and JAK2 tyrosine phosphorylation. Taken together, these results suggest that biologic activities of GM-CSF in vascular endothelium may, in part, be elicited by GM-CSFR-mediated JAK2 activation.

Chemokines are key molecules in directing leukocyte migration toward sites of inflammation. We have previously cloned a putative CC chemokine receptor gene, TER1, whose expression is restricted to lymphoid tissues and cell lines. Recently, this receptor has been shown to signal in response to the human CC chemokine I-309 and thus it has been renamed CCR8 according to the current nomenclature. In the present study, we report the identification of the CC chemokines thymus and activation-regulated cytokine (TARC) and macrophage inflammatory protein-1 beta (MIP-1 beta) as CCR8 ligands, as they induce chemotaxis in CCR8 Jurkat stable transfectants. Furthermore, we have generated a polyclonal antiserum that is able to recognize the CCR8 molecule in transfectant lysates. The pattern of CCR8 mRNA expression and the functional effects exerted by its ligand suggest that the triggering of this receptor may regulate multiple functions including activation, migration and proliferation of lymphoid cells.

Upon exposure to immune or inflammatory stimuli, dendritic cells (DC) migrate from peripheral tissues to lymphoid organs, where they present antigen. The molecular basis for the peculiar trafficking properties of DC is largely unknown. In this study, mouse DC were generated from CD34+ bone marrow precursors and cultured with granulocyte-macrophage-CSF and Flt3 ligand for 9 days. Chemokines active on immature DC include MIP1alpha, RANTES, MIP1beta, MCP-1, MCP-3, and the constitutively expressed SDF1, MDC, and ELC. TNF-alpha-induced DC maturation caused reduction of migration to inducible chemokines (MIP1alpha, RANTES, MIP1beta, MCP-1, and MCP-3) and increased migration to SDF1, MDC, and ELC. Similar results were obtained by CD40 ligation or culture in the presence of bacterial lipopolysaccharide. TNF-alpha down-regulated CC chemokine receptor (CCR)1, CCR2, and CCR5 and up-regulated CCR7 mRNA levels, in agreement with functional data. This study shows that selective responsiveness of mature and immature DC to inducible vs. constitutively produced chemokines can contribute to the regulated trafficking of DC.

Toll is a Drosophila gene essential for ontogenesis and anti-microbial resistance. Several orthologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLRs). Human TLRs are a growing family of molecules involved in innate immunity. TLRs are characterized structurally by a cytoplasmic Toll/interleukin-1 receptor (TIR) domain and by extracellular leucine-rich repeats. TLRs characterized so far activate the MyD88/interleukin-1 receptor-associated kinase (IRAK) signalling pathway. Genetic, gene-transfer and dominant-negative approaches have involved TLR family members (TLR2 and TLR4) in Gram-positive and Gram-negative bacteria recognition and signalling. Accumulating evidence suggests that TLR2 is also involved in signalling-receptor complexes that recognize components of yeast and mycobacteria. However, the definitive roles of other TLRs are still lacking. A systematic approach has been used to determine whether different human leucocyte populations selectively or specifically express TLR mRNA. Based on expression pattern, TLR can be classified as ubiquitous (TLR1), restricted (TLR2, TLR4 and TLR5) and specific (TLR3). Expression and regulation of distinct but overlapping ligand-recognition patterns may underlie the existence of a large, seemingly redundant TLR family. Alternatively, the expression of a TLR in a single cell type may indicate a specific role for this molecule in a restricted setting.

Phagocytes infiltrating neoplastic tissues have peculiar membrane phenotype and functional properties. Tumor-associated macrophages (TAM) play a complex, ambiguous role in the regulation of primary tumor growth and metastasis (a "macrophage balance"). Yet these cells are strategically located at the very interface between tumor and host and represent a potential target for immunomodulation. A better understanding of the regulation and function of TAM may provide a less empirical basis of or rational design of therapeutic approaches, as vividly illustrated by the antitumor activity of i.p. in IFN ovarian cancer patients with minimal residual disease resistant to chemotherapy.