A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.

Tumor-specific and non-specific CD3+, TcR alpha beta+, CD8+ cytotoxic T-cell (CTL) clones, isolated from tumor-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) of a melanoma patient and allogeneic LAK cells, were used to investigate the requirements for bypassing the low lysability of some melanoma clones derived from an s.c. metastasis from which highly lysable clones were also obtained. Cytofluorimetric analysis showed that all melanoma clones expressed ICAM-1, although to different extents, reaching a 10-fold difference in fluorescence units, while HLA class-I antigens were similarly expressed. The differences in expression of ICAM-1 among tumor clones correlated with differences in lysability, by both specific and non-specific CTL, but were not large enough to affect lymphocyte-tumor conjugate formation. Cytokine- or gene-transfer-mediated up-regulation of ICAM-1 did not induce de novo lysis of ICAM-1low tumor cells; however, it markedly enhanced a low level of killing of the same cells by tumor-specific, TcR-dependent and HLA-restricted CTL clones but not by non-specific, TcR-independent effectors. In addition, lysis of melanoma clones by any effector was similarly inhibited by anti-ICAM-1 and anti-LFA-1 antibodies. This indicates that by-pass of low lysability of ICAM-1low melanoma clones by CTL clones, after ICAM-1 up-regulation, is possible only if simultaneous LFA-1 and TcR engagement takes place. In addition, these results suggest that the constitutive high level of expression of ICAM-1 on the subset of ICAM-1high melanoma cells must be only one of the factors contributing to the high lysability of these cells by any effector.

We studied cytokine-related functional properties of four mouse endotheliomas from different anatomical sites obtained by transformation with middle T oncogene. We examined mRNA expression of IL-6, IL-1 alpha, macrophage-CSF, granulocyte/macrophage-CSF, and two members of an emerging super-family of chemotactic cytokines (JE/monocyte chemoattractant protein-1 (MCP-1) and KC). Exposure to IL-1 augmented or induced cytokine gene transcripts in three endothelioma lines (eEnd.1, sEnd.1, and tEnd) with maximal expression in tEnd.1 cells. Endothelioma cells also responded to TNF-alpha and LPS. Levels of IL-6 and monocyte chemotactic activity (a JE/MCP activity) correlated with mRNA expression. IL-1 also induced production of procoagulant activity and platelet-activating factor in endothelioma cells, with heterogeneity in the levels of response among individuals lines. Murine melanoma B16-F1, human colon carcinoma HT29 cells, CB33MT lymphoblastoid cells, and monocytes adhered to endothelioma monolayers and the adhesive properties of these cell lines were modulated by IL-1 beta, with marked differences among themselves. Murine EC derived from brain capillaries, used as control, shared several properties with bEnd.4 line. Endothelioma lines cause tumors by recruiting host cells. The capacity to produce cytokines that directly or indirectly attract host vascular cells, may play an important role in hemangioma induction in vivo. Murine endothelioma lines, generated by transformation with the polyoma middle T oncogene, retain functional properties of normal endothelium, and may represent an invaluable tool for analysis of the immunobiology and heterogeneity of EC in different tissues.

Tumor-derived chemotactic factors (TDCF) have been identified and thought to play a role in the regulation of macrophage infiltration in neoplastic tissues. The present study was designed to assess the in vivo relevance of the TDCF molecularly identified as monocyte chemotactic protein/JE, by investigating murine sarcoma clones expressing different levels of MCP/JE. The 1D3 clone derived from the B77 RSV-induced sarcoma expressed appreciable levels of MCP/JE mRNA and, concomitantly, chemotactic activity for mononuclear phagocytes. In contrast, the 5B11 clone from the same tumor had undetectable levels of MCP/JE transcripts and little or no chemotactic activity. The chemotactic activity of 1D3 cells was blocked by an appropriate specific antiserum. The in vitro growth rate of the 2 sarcoma lines was identical. Upon in vivo transplantation, the 1D3 clone showed a substantially higher level of tumor-associated macrophages (28.9%; range 21%-34%) than the 5B11 clone (16.6%; range 13%-20%). 5B11-induced tumors appeared earlier and grew faster than those induced by 1D3. The difference in growth rate and in macrophage infiltration between 1D3 and 5B11 clones was also evident upon transplantation into nude mice. These results are consistent with the hypothesis that TDCF, identified as MCP/JE, is one important determinant of macrophage infiltration in tumors.

Recombinant monocyte-chemotactic and activating factor (rMCAF; alternative acronyms MCP-1, TDCF, human JE) induced migration of human monocytes across polycarbonate or nitrocellulose filters. Maximal induction of migration was observed at a concentration of 10 ng/ml (10(-9) M). Checkerboard analysis revealed that rMCAF elicited true gradient-dependent chemotactic migration, although a gradient independent chemokinetic effect was observed at low concentrations (1-5 ng/ml). rMCAF caused a rapid (less than 5 s) and transient (approximately 1.5 min) increase of free cytosolic Ca2+ ions, as assessed by the fura-2 probe. No Ca2+ increase was detected in neutrophils or lymphocytes stimulated by rMCAF. Studies conducted in the absence of extracellular Ca2+ or in the presence of Ni2+ (an inhibitor of Ca2+ influx) suggested that the increase of intracellular Ca2+ induced by rMCAF is dependent on the influx of extracellular Ca2+ through plasma membrane channels. Bordetella pertussis toxin inhibited the intracellular Ca2+ elevation and chemotaxis caused by rMCAF. The possible involvement of Ca(2+)-dependent protein kinases in rMCAF signaling pathway(s) was explored using inhibitors. Inhibitors of GMP-dependent kinase and myosin L chain kinase had no effect on rMCAF-induced monocyte migration. In contrast, protein kinase C/cAMP-dependent kinase inhibitors (such as, C-I, H-7, HA-1004, KT5720, and Staurosporine) markedly decreased rMCAF induced chemotaxis suggesting the involvement of a serine/threonine protein kinase, possibly protein kinase C, in rMCAF signaling pathway.

Tumor-associated macrophages (TAM) isolated from two murine sarcomas (mFS6 and MN/MCA1) had high levels of proliferative activity (7 to 11% of cells in S phase) compared to peritoneal macrophages (1 to 2% of cells in S phase). In an effort to elucidate the mechanisms responsible for the proliferative activity of TAM, expression of c-fms and macrophage (M)-CSF was investigated in TAM and sarcoma cells. TAM had high levels of mRNA transcripts of the c-fms protooncogene, which encodes a tyrosine kinase probably identical to the M-CSF receptor, but did not express M-CSF transcripts whereas sarcoma cells had high levels of M-CSF mRNA. Sarcoma cell conditioned medium had M-CSF activity on bone marrow cells and induced proliferation of peritoneal exudate and bone marrow-derived macrophages. These activities were blocked by anti-M-CSF antibodies. These findings outline a paracrine circuit in the regulation of TAM proliferation, involving M-CSF, secreted by sarcoma cells and acting on c-fms expressing TAM. Inasmuch as TAM from these murine sarcomas have tumor growth promoting activity, a "ping pong" reciprocal feeding interaction may occur between macrophages and neoplastic cells in these tumors.

Using a differential screening strategy, clone 133 was identified as an immediate early gene induced by interleukin-1 (IL-1) in human endothelial cells (HEC). The sequence of the cDNA revealed identity with a recently identified member of the inhibitors of nuclear factor kB family (IkB). IkB/clone 133 is expressed at low levels in unstimulated endothelial cells and is rapidly induced by the inflammatory cytokines IL-1 and tumor necrosis factor α(TNFα*), but not interleukin-6. Inducibility by IL-1 is abolished by the IL-1 receptor antagonist. Cycloheximide superinduces IkB/clone 133 expression in HEC. IL-1 and TNFα have little or no effect on IkB/clone 133 expression in a fibrosarcoma cell line responsive to these polypeptide mediators. These results show for the first time cytokine-inducible lineage-specific expression of a member of the IkB family.

Primary cultures of mouse embryonic fibroblasts and confluent monolayers of mouse fibroblastoid cells (L929) were found to secrete a chemotactic factor specific for monocytes. It biological activity was deduced from both the migration distance under agarose and the number of migrated monocytes in the micropore filter method. The monocyte chemotactic protein (MCP) was inducible in these cells by double-stranded RNA and by infection with virus. In embryonic fibroblasts MCP was also produced in response to the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Under all conditions for induction of MCP tested no production of chemotactic activity for granulocytes could be detected. MCP activity from virally infected L929 cells was concentrated and purified by sequential adsorption to controlled pore glass, heparin-Sepharose chromatography, ion-exchange FPLC and reversed-phase HPLC. Pure MCP was found to occur mainly as a 7-8-kDa protein. Although the mature protein possessed a blocked NH2-terminus, it was identified by enzymatic cleavage and sequence analysis of an internal fragment. The sequence obtained corresponded to a part of the cDNA-derived protein sequence of the murine 'competence' (JE) gene, inducible in fibroblasts by cytokines and virus. In all probability the 7-8-kDa MCP form represents the natural product of the mouse gene JE. Murine MCP can thus be classified in the novel family of small inducible inflammatory proteins.

Receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL) signaling promotes mammary tumor development in experimental models. Circulating concentrations of soluble RANKL (sRANKL) may influence breast cancer risk via activation of RANK signaling; this may be modulated by osteoprotegerin (OPG), the decoy receptor for RANKL. sRANKL and breast cancer risk by hormone receptor subtype has not previously been investigated. A case-control study was nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. This study included 1,976 incident invasive breast cancer cases [estrogen receptor positive (ER+), <i>n</i> = 1,598], matched 1:1 to controls. Women were pre- or postmenopausal at blood collection. Serum sRANKL was quantified using an ELISA, serum OPG using an electrochemiluminescent assay. Risk ratios (RR) and 95% confidence intervals (95% CI) were calculated using conditional logistic regression. Associations between sRANKL and breast cancer risk differed by tumor hormone receptor status (<i>P</i>_het = 0.05). Higher concentrations of sRANKL were positively associated with risk of ER+ breast cancer [5th vs. 1st quintile RR 1.28 (95% CI, 1.01-1.63); <i>P</i>_trend = 0.20], but not ER- disease. For both ER+ and estrogen and progesterone receptor positive (ER+PR+) breast cancer, results considering the sRANKL/OPG ratio were similar to those for sRANKL; we observed a suggestive inverse association between the ratio and ER-PR- disease [5th vs. 1st quintile RR = 0.60 (0.31-1.14); <i>P</i>_trend = 0.03]. This study provides the first large-scale prospective data on circulating sRANKL and breast cancer. We observed limited evidence for an association between sRANKL and breast cancer risk. <i>Cancer Prev Res; 10(9); 525-34. ©2017 AACR</i>.

Previous case-control studies have suggested a possible increased risk of Amyotrophic Lateral Sclerosis (ALS) with physical activity (PA), but this association has never been studied in prospective cohort studies. We therefore assessed the association between PA and risk of death from ALS in the European Prospective Investigation into Cancer and Nutrition. A total of 472,100 individuals were included in the analysis, yielding 219 ALS deaths. At recruitment, information on PA was collected thorough standardised questionnaires. Total PA was expressed by the Cambridge Physical Activity Index (CPAI) and analysed in relation to ALS mortality, using Cox hazard models. Interactions with age, sex, and anthropometric measures were assessed. Total PA was weakly inversely associated with ALS mortality with a borderline statistically significant trend across categories (p = 0.042), with those physically active being 33% less likely to die from ALS compared to those inactive: HR = 0.67 (95% CI 0.42-1.06). Anthropometric measures, sex, and age did not modify the association with CPAI. The present study shows a slightly decreased-not increased like in case-control studies-risk of dying from ALS in those with high levels of total PA at enrolment. This association does not appear confounded by age, gender, anthropometry, smoking, and education. Ours was the first prospective cohort study on ALS and physical activity.

The present study was designed to define molecules and structures involved in the interaction of natural killer (NK) cells with the vascular endothelium in vitro. Resting and interleukin 2 (IL-2)-activated NK cells were studied for their capacity to adhere to resting and IL-1-treated human umbilical vein endothelial cells (EC). In the absence of stimuli, NK cells showed appreciable adhesion to EC, with levels of binding intermediate between polymorphs and monocytes. The binding ability was increased by pretreatment of NK cells with IL-2. Using the appropriate monoclonal antibody, the beta 2 leukocyte integrin CD18/CD11a was identified as the major adhesion pathway of NK cells to unstimulated EC. Activation of EC with IL-1 increased the binding of NK cells. In addition to the CD18-CD11a/intercellular adhesion molecule pathway, the interaction of resting or IL-2-activated NK cells to IL-1-activated EC involved the VLA-4 (alpha 4 beta 1)-vascular cell adhesion molecule 1 receptor/counter-receptor pair. No evidence for appreciable involvement of endothelial-leukocyte adhesion molecule was obtained. Often, NK cells interacted either with the culture substrate or with the EC surface via dot-shaped adhesion structures (podosomes) protruding from the ventral surface and consisting of a core of F-actin surrounded by a ring of vinculin and talin. The identification of molecules and microanatomical structures involved in the interaction of NK cells with EC may provide a better understanding of the regulation of NK cell recruitment from blood, their extravasation, and their migration to tissues.

Leucocytes and vascular cells interact closely in inflammation and immunity and cytokines are important mediators of this interaction. The present study was designed to define the capacity of human endothelial cells (HEC) to produce a monocyte-derived neutrophil chemotactic factor (provisionally termed IL-8). IL-8 is a polypeptide chemotactic for neutrophils originally identified in the culture supernatant of lipopolysaccharide (LPS)-stimulated monocytes. IL-1 induced high levels of production of neutrophil chemotactic activity in culture supernatants of HEC. Optimal stimulation of activity was observed when HEC were cultured with 10-100 ng/ml IL-1 beta for 16 hr. Anti-IL-8 antibody blocked the chemotactic activity for neutrophils of IL-1-activated HEC supernatants. IL-1-treated HEC expressed high levels of IL-8 mRNA transcripts, as assessed by Northern blot analysis. Tumour necrosis factor (TNF) and LPS, unlike the inflammatory monokine IL-6, also induced IL-8 expression. Nuclear run-off experiments revealed that IL-1 activated transcription of the IL-8 gene. The production of IL-8 may represent a mechanism whereby endothelial cells, exposed to inflammatory signals, participate in the regulation of neutrophil extravasation.