Amalia Kinarty

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.

Acute release of cytokines is proportional to tissue injury induced by surgical trauma and shock in rats.

Cytokines are important mediators of the inflammatory reaction and microvascular injury after trauma and tissue ischemia. The plasma activity of a cytokine reflects the net effect of positive and negative signals. We examined the sequential serum activity of IL-1, IL-2, IL-6, and TNF in a severe model of splanchnic artery occlusion (SAO) shock induced in rats by total occlusion of the superior mesenteric and the celiac arteries for 40 min. A control group with negligible surgical intervention and two sham-shock groups, one with minor operation and another with major surgery employed in SAO rats, both without vascular occlusion, were also studied. No IL-1 activity was detected throughout the 190-min experimental protocol in any of the groups. Low activity of IL-2 was measured only in SAO rats (∼1 U/ml at the peak). We found graded increases in serum TNF and IL-6 activities which were proportional to the surgical trauma and were highest in SAO rats (IL-6 up to 30 U/ml,P<0.01 from both sham groups; TNF, 2500 pg/ml 30 min after reperfusion,P<0.01 from both sham groups). These data further support the role played by cytokines in the early mediation of surgical trauma and shock.

Exposure to hyperbaric oxygen induces tumour necrosis factor‐alpha (TNF‐α) secretion from rat macrophages

We investigated the secretion of TNF‐α by monocytes and macrophages derived from the peripheral blood, spleen, and lungs after a single exposure to a therapeutic profile of hyperbaric oxygen (HBO). Rats were exposed for 90 min to either 100% oxygen at 0 28 MPa (2–8 atmospheres absolute) or air. Immediately after exposure, mononuclear cells were isolated from blood, spleen, and lungs and cultured for 18h. The secretion of TNF‐α from the cultured monocytes/macrophages was determined with and without stimulation with lipopolysaccharide (LPS). Exposure to hyperbaric oxygen induced a significant increase in the spontaneous ex vivo secretion of TNF‐α (without LPS) by mononuclear cells from the blood, spleen, and lung (P < 0 05 from air controls). Stimulation with LPS after exposure to HBO induced a significant increase in TNF‐α secretion by lung and spleen macrophages compared with air controls (P<005). However, absolute TNF‐α levels were not significantly higher than those achieved ‘spontaneously’ in macrophages exposed to HBO without LPS. Stimulation with LPS induced a marked increase in secretion of TNF‐α from blood monocytes after exposure to air, but not after exposure to HBO. These results provide evidence in support of a role played by TNF‐α in mediating HBO effects on different tissues and their immune responses.

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.

Divergent effects of cytokines on human leukocyte antigen‐DR antigen expression of neoplastic and non‐neoplastic human thyroid cells

Apparently complex modulatory effects of alpha‐interferon (α ‐IFN), tumor necrosis factor (TNF), and epidermal growth factor (EGF) have been found in neoplastic human thyroid cells, which could possibly affect the final outcome in neoplastic disease. This was achieved by examining the influence of α‐IFN, TNF, and EGF alone and in combination, on human leukocyte antigen‐DR (DR) antigen expression and viability of neoplastic and non‐neoplastic human thyroid cells in culture. α‐IFN‐induced DR antigen expression on non‐neoplastic human thyroid cells, whereas TNF‐α or EGF alone were ineffective. The addition of the same TNF‐α concentrations (10 to 100 ng/ml) to α‐IFN enhanced the expression of DR antigens compared with the effect of α‐IFN alone. However, EGF inhibited α‐IFN‐induced DR on the same cells and at the same concentrations (10 to 500 ng/ml) at which the growth factor alone was ineffective. In contrast to the common pattern of cytokine effects on DR expression of all nonmalignant thyroid cell lines, neoplastic thyroid cell lines showed divergent responses to α‐IFN, TNF‐α, and EGF. In three malignant thyroid cell lines that were DR negative (follicular carcinoma WRO 82‐1 and NRO 87‐1 cell lines, and anaplastic carcinoma ARO 81‐1), DR antigen could be induced by α‐IFN and enhanced by TNF‐α, whereas EGF was ineffective. In a fourth cell line (an anaplastic carcinoma SW1736) α‐IFN, TNF‐α, and EGF alone were capable of inducing DR, and a combination of either TNF‐α and EGF with α‐IFN potentiated DR induction. In a fifth neoplastic cell line (papillary carcinoma, NPA) that constitutively expressed surface DR, its expression was inhibited by both α‐IFN and TNF‐α and was not affected by EGF.

Hypoxia enhances lysosomal TNF-α degradation in mouse peritoneal macrophages

Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor-alpha (TNF-alpha) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O(2) < 0.3%) reduces the secretion of LPS-induced TNF-alpha (P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF-alpha mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF-alpha by twofold (P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF-alpha by twofold (P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF-alpha in hypoxic peritoneal macrophages.

Increased spontaneous secretion of IL-6 from B cells of patients with B chronic lymphatic leukaemia (B-CLL) and autoimmunity

We studied B cells from 18 patients with B‐CLL, six of them with autoimmune haemolytic anaemia, for spontaneous secretion of IL‐6. Our aim was to determine whether the increased incidence of autoimmune disease found in B‐CLL patients is associated with enhanced spontaneous IL‐6 secretion. IL‐6 was measured by the effect of B cell supernatants on the proliferation of an IL‐6 dependent plasmacytoma cell line T1165. The highest IL‐6 values (7.4±1.8 U/ml) were measured in supernatants derived on day 3 of culture from lymphocytes of the six patients with B‐CLL and concomitant autoimmune disease. The maximal IL‐6 values for 10 patients with B‐CLL only were 2.8±0.3 U/ml and for 10 age‐matched controls, 0.8±0.3 U/ml (P < 0.01, each group compared with the other). We conclude that there is an association between B‐CLL, autoimmune disease and the spontaneous in vitro secretion of IL‐6. Further studies are needed to determine whether the IL‐6 secretion plays a role in the pathogenesis of autoimmune disease in patients with B‐CLL.

Interferon-β and -γ, but not tumor necrosis factor-α, demonstrate immunoregulatory effects on carcinoma cell lines infected with human papillomavirus

Mechanisms whereby cells infected with human papillomavirus (HPV) escape immune surveillance, ultimately leading to invasive cervical carcinoma, may involve changes in local cytokine production, loss of responsiveness to cytokines, and alterations in the expression of immune‐regulatory molecules such as histocompatibility‐related leukocyte antigen (HLA) Class 1 and 2 and ICAM‐I. This study examined the separate and combined effects of immune‐activating cytokines, interferon (IFN)‐γ, IFN‐β, and tumor necrosis factor (TNF)‐α, on the expression of these molecules.

Divergent effects of ischemia/reperfusion and nitric oxide donor on TNFalpha mRNA accumulation in rat organs.

We previously showed that serum TNFalpha bioactivity in rats is proportional to the extent of graded tissue injury caused by laparotomy, intestinal ischemia, and reperfusion and that the spleen is an important source of TNFalpha secretion in this condition. TNFalpha production varies, depending on the type and duration of tissue injury. It is also affected by other mediators, such as nitric oxide (NO). TNFalpha is known to increase NO production, but the effect of NO on the production of TNFalpha has not yet been fully elucidated. In this study we determined the levels of TNFalpha mRNA in rat organs after graded injury caused by anesthesia, laparotomy, intestinal ischemia, and reperfusion and evaluated the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on it. Samples from different organs were removed, and TNFalpha gene expression was evaluated by semiquantitative RT-PCR. TNFalpha mRNA was not detected in the intestine (the ischemic organ) and in the kidney, brain, heart, or liver after all 4 experimental protocols. In the mesenteric lymph node (draining the ischemic organ) a basal level of expression of TNFalpha mRNA was detected in the control (anesthesia alone) group, which was increased significantly after ischemia. In the spleen (a remote immune organ not directly involved in the ischemia), a significant gradual increase in TNFalpha mRNA, which correlated to the severity of the experimental protocol, was observed. In the lung (a central participant in post-injury multiple organ failure), all interventions increased TNFalpha mRNA. Infusion of SNAP exerted a differential effect on TNFalpha mRNA: diminished its accumulation in the lymph node, enhanced it in the lung, and had no effect in the spleen. The divergent organ pattern of TNFalpha transcription emphasizes the importance of its localized expression, which is critical to the understanding of its autocrine and paracrine actions in ischemia and reperfusion.