Michal A. Rahat

The Role of Macrophage-Derived IL-1 in Induction and Maintenance of Angiogenesis

Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

Transforming growth factor-β suppresses tumor necrosis factor α-induced matrix metalloproteinase-9 expression in monocytes

The inflammatory response is marked by the release of several cytokines with multiple roles in regulating leukocyte activities, including the secretion of matrix metalloproteinases (MMPs). Although the effects of individual cytokines on monocyte MMP expression have been studied extensively, few studies have examined the influence of combinations of cytokines, which are likely present at inflammatory sites. Herein, we report our investigation of the combinatorial effects of tumor necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β on MMP‐9 synthesis. We found that TGF‐β suppressed TNF‐α‐induced MMP‐9 secretion by MonoMac‐6 monocytic cells in a dose‐dependent manner, with a maximal effect of TGF‐β observed at 1 ng/ml. Such suppression was likely regulated at the pretranslational level, because steady‐state mRNA levels of TNF‐α‐induced MMP‐9 were reduced by TGF‐β, and pulse‐chase radiolabeling also showed a decrease in new MMP‐9 protein synthesis. The suppressive effects of TGF‐β were time dependent, because short exposures to TNF‐α before TGF‐β or simultaneous exposure to both cytokines efficiently reduced MMP‐9 secretion. Expression of the tissue inhibitor of metalloproteinases (TIMP)‐1 and TNF‐α receptors was unaffected by either cytokine individually or in combination. Affinity binding with radiolabeled TGF‐β demonstrated that levels of TGF‐β receptors were not increased after preincubation with TGF‐β. Suppression of TNFα‐induced MMP‐9 secretion by TGF‐β correlated with a reduction in prostaglandin E2 (PGE2) secretion. Furthermore, the effect of TGF‐β or indomethacin on blockage of TNF‐α‐stimulated MMP‐9 production was reversed by the addition of either exogenous PGE2 or the cyclic AMP (cAMP) analogue Bt2cAMP. Thus, we concluded that TGF‐β acts as a potent suppressor of TNF‐α‐induced monocyte MMP‐9 synthesis via a PGE2‐ and cAMP‐dependent mechanism. These results suggest that various combinations of cytokines that are present at inflammatory sites, as well as their balance during different stages of inflammation, may provide the signals necessary for directing MMP‐mediated leukocyte activities.

Hypoxia Inactivates Inducible Nitric Oxide Synthase in Mouse Macrophages by Disrupting Its Interaction with α-Actinin 4

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-γ induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 ± 14 μM, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 ± 1.7 μM, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and α-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.

Hypoxia reduces the output of matrix metalloproteinase-9 (MMP-9) in monocytes by inhibiting its secretion and elevating membranal association

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase‐9 (MMP‐9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor α (TNF‐α)]. We show that hypoxia (<0.3% O2 for 48 h) reduced the output of TNF‐α‐induced proMMP‐9 by threefold (P<0.01) in the U937 monocytic cell line and in primary human monocytes. TNF‐α induced MMP‐9 transcription by threefold, but no significant difference was observed in MMP‐9 mRNA steady‐state between normoxia and hypoxia, which inhibited the trafficking of proMMP‐9 via secretory vesicles and increased the intracellular accumulation of proMMP‐9 in the cells by 47% and 62% compared with normoxia (P<0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP‐9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP‐9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP‐9. Thus, hypoxia post‐translationally reduced the secreted amounts of proMMP‐9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

Fibronectin‐bound TNF‐α stimulates monocyte matrix metalloproteinase‐9 expression and regulates chemotaxis

Tumor necrosis factor α (TNF‐α) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF‐α avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF‐α complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM‐bound TNF‐α by examining its effects on MMP‐9 secretion by monocytes. Immunofluorescent staining indicated that LPS‐activated monocytes deposited newly synthesized TNF‐α into ECM‐FN. FN‐bound TNF‐α (FN/TNF‐α) significantly up‐regulated MMP‐9 expression and secretion by the human monocytic cell line MonoMac‐6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF‐α activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Chemotaxis through ECM gels in the presence of soluble or bound TNF‐α was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac‐6 cells to FN/TNF‐α required functional activated β1 integrins, FN/TNF‐α‐induced MMP‐9 secretion was independent of binding to β1 integrins, since MMP‐9 secretion was unaffected by: (1) neutralizing mAb to α4, α5, and β1 subunits, which blocked cell adhesion; (2) a mAb that stimulated β1 integrin‐mediated adhesion; and (3) binding TNF‐α to the 30‐kDa amino‐terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell‐adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM‐degrading enzymes. These TNF‐α‐ECM interactions may serve to limit the cytokine’s availability and bioactivity to target areas of inflammation.

Etiology of cervical cancer: current concepts.

The data on the etiological factors presented here may enable us to suggest a synergism between the various factors associated with the pathogenesis of cervical cancer. Infection of the cervix by HPV 16/18 may result in persistence of viral DNA. The persistent HPV-DNA undergoes disruption at the E2 region, when integrated into the host genome. The transcriptional products E6 and E7 oncoproteins bind to and cause the degradation of p53 and Rb tumor-suppressor gene products. It is possible that, at that point, other cofactors may be involved in the progression toward a precancerous or cancerous condition. Those cofactors may include cigarette smoking, by introducing co-carcinogens to the tissue or by suppressing the local or systemic immune resistance similar to the effect of depressed immune resistance seen in AIDS or immunosuppression of transplant patients; hormones, by enhancing growth of HPV and transformation of HPV infected cells; low serum vitamin levels leading to decreased tissue resistance; or other infections causing local inflammation and the production of free radicals. CIN develops, leading eventually to cervical cancer.

Hypoxia reduces CD80 expression on monocytes but enhances their LPS‐stimulated TNF‐α secretion

Monocytes/macrophages in ischemic tissues are involved in inflammation and suppression of adaptive immunity via secretion of proinflammatory cytokines and reduced ability to trigger T cells, respectively. We subjected human mononuclear cells and mouse macrophages to hypoxia and reoxygenation, the main constituents of ischemia and reperfusion, and added lipopolysaccharide (LPS) to simulate bacterial translocation, which frequently accompanies ischemia. We monitored the secretion of tumor necrosis factor α (TNF‐α) and the surface expression of human leukocyte antigen‐DR and the costimulatory molecules CD80 and CD86 on monocytes/macrophages. Hypoxia selectively reduced the surface expression of CD80 (P<0.01), and synergistically with LPS, it enhanced TNF‐α secretion (P<0.003). Reoxygenation reversed both phenomena. In the mouse macrophage cell line RAW 264.7, hypoxia reduced the surface expression of CD80 and increased its concentrations in the supernatants (P<0.01). Down‐regulation of the mRNA coding for the membrane‐anchored CD80 was observed, suggesting that hypoxia triggers alternative splicing to generate soluble CD80. Cumulatively, these results suggest that hypoxia simultaneously affects monocytes/macrophages to enhance inflammation and reduce their ability to initiate adaptive‐immunity responses associated with ischemic injury.

Telomerase activity and cytokeratin 20 as markers for the detection and followup of transitional cell carcinoma: an unfulfilled promise.

PURPOSE Telomerase activity compensates for the erosion of chromosomes and it has been detected in a wide variety of human tumors. Cytokeratin 20, an intermediate filament of epithelial cells, is expressed particularly in the urinary tract. These 2 molecules are candidates to become markers for the detection and followup of bladder carcinoma. We evaluate whether each molecule may serve as a potential marker and whether the 2 combined would improve the detection or followup of bladder carcinoma in a noninvasive manner. MATERIALS AND METHODS We obtained 44 morning urine samples from patients with transitional cell carcinoma patients and 26 from age matched patients with a wide variety of clinical disorders but no malignancy of any kind. A telomerase polymerase chain reaction-enzyme-linked immunosorbent assay kit was used to determine telomerase activity and cytokeratin 20 expression was determined by nested reverse transcriptase-polymerase chain reaction. RESULTS All samples tested positive for cytokeratin 8 expression, which verified epithelial cells in the urine samples. Of the 44 transitional cell carcinoma cases of all stages and grades 37 (84.1%) were positive for telomerase activity, 36 (81.8%) were positive for cytokeratin 20 expression and 65.9% were double positive. Of the 29 controls with various clinical conditions other that malignancy 22 (75.9%) were positive for telomerase activity, 13 (44.83%) were positive for cytokeratin 20 expression and 34.6% were double positive. CONCLUSIONS Telomerase activity and cytokeratin 20 expression are not specific for malignancy and may be detected in many nonmalignant pathological conditions. Therefore, their use as potential markers of bladder carcinoma should be carefully reevaluated.

Native and fragmented fibronectin oppositely modulate monocyte secretion of MMP-9

Monocytes remodel the extracellular matrix (ECM) by secreting proteins composing the ECM such as fibronectin (FN) and degrading proteases such as matrix metalloproteinase‐9 (MMP‐9), which cleaves FN into fragments. The effects of FN and its fragmented products on the expression of monocyte MMP‐9 are controversial and largely unknown. We showed that in human monocytes, the proinflammatory cytokine TNF‐α induced MMP‐9 secretion and increased fragmentation of FN into distinct fragments. When primary monocytes or the U937 monocytic cell line were incubated on a plastic substrate, plastic‐coated with native FN, and plastic‐coated with fragmented FN (frag‐FN), native FN inhibited TNF‐α‐induced proMMP‐9 secretion by twofold (P<0.01) compared with plastic or frag‐FN. Exploration of the dynamics of inflammation by incubating cells sequentially on the three substrates showed that frag‐FN opposed the inhibitory effect of native FN. Inhibition of proMMP‐9 by native FN was exerted at the translational level, as no change in MMP‐9 mRNA, intracellular protein accumulation, or proteomic degradation was observed, and when degradation was blocked, no de novo translation of MMP‐9 could be measured. We also showed that the reduction of MMP‐9 secretion by native FN was responsible for attenuated migration of U937 cells (P<0.05). We suggest that in the inflammatory tissue, intact, native FN has a homeostatic role in harnessing MMP‐9 activity. However, as fragmented products accumulate locally, they alleviate the inhibition and enable faster migration of the monocytes through the degraded ECM.

Telomerase activity in patients with transitional cell carcinoma

Telomerase activity is not detectable in normal cells, and their telomers shorten until the chromosome is unable to replicate. Immortal cells have short but stable chromosomes and increased telomerase activity. Transitional cell carcinoma (TCC) has only a few useful markers of diagnostic or prognostic importance. The objective of this study was to determine whether there was a correlation between telomerase activity and the grade or stage of TCC, and whether the enzymes activity could serve as a biochemical marker of this tumor.