Interleukins 6/8 and cyclooxygenase-2 release and expressions are regulated by oxidative stress-JAK2/STAT3 signaling pathway in human bronchial epithelial cells exposed to particulate matter ≤2.5 µm.

Atmospheric particulate matter with a diameter ≤2.5 µm (PM2.5) can induce inflammation of the respiratory system, which is the pathological basis of asthma or other respiratory diseases; however, the underlying regulation mechanism has not been clearly addressed. The aim of this study was to explore the potential role of the oxidative stress-JAK/STAT signaling pathway in the inflammation of human bronchial epithelial cells induced by PM2.5. The human bronchial epithelial cell line 16HBE cells were stimulated with PM2.5 at 50 and 100 µg/mL doses for 12 or 24 hours. Intracellular reactive oxygen species (ROS) was detected using flow cytometry. Gene and protein expressions of JAK2, STAT3 and cyclooxygenase 2 (COX-2) were determined using reverse transcription-polymerase chain reaction and western blotting, respectively. The ratio of intracellular glutathione/glutathione disulfide (GSH/GSSG) and the levels of interleukin (IL)-6 and IL-8 in cellular supernatant were analyzed using enzyme-linked immunosorbent assay. The results indicated that PM2.5 treatment significantly increased gene expressions of JAK2/STAT3 and protein levels of p-JAK2/p-STAT3, accompanied by increased intracellular ROS levels, decreased GSH/GSSG ratio at 50 and 100 µg/mL of PM2.5, and significantly enhanced levels of IL-6, IL-8 and COX-2 at a dose of 100 µg/mL. Pretreatment with N-acetyl-l-cysteine (NAC) attenuated the oxidative stress induced by PM2.5; similarly, pretreatment with AG490 (an inhibitor of JAK) decreased the cytokine levels stimulated by PM2.5. Therefore, we concluded that PM2.5 exposure could activate oxidative stress-JAK2/STAT3 signaling pathway, elevate the levels of IL-6, IL-8 and COX-2 in 16HBE cells, which can be inhibited by the NAC or AG490.

Fully human anti-interleukin-8 monoclonal antibodies: potential therapeutics for the treatment of inflammatory disease states.

Interleukin-8 (IL-8) is a potent chemotactic cytokine implicated in the pathogenesis of a number of inflammatory disease states. Agents that block the binding of IL-8 to its receptor have been shown to block inflammation in animal models of disease. This suggests that drugs specifically targeting IL-8 may prove efficacious in treating multiple human diseases. To this end, we developed a panel of fully human anti-IL-8 monoclonal antibodies (mAbs). These human antibodies were generated from XenoMouse strains, mice created by introducing megabase-size unrearranged human immunoglobulin heavy and kappa light chain loci into a mouse genome in which the corresponding endogenous loci have been inactivated. From the panel of more than 50 mAbs, two antibodies, K4.3 and K2.2, were further characterized and evaluated for their specificity, productivity, affinity, and biological activity. Both K4.3 and K2.2 bind human IL-8 with high affinity (Kd of K4.3 = 2.1x10(10) M; Kd of K2.2 = 2.5x10(-10) M). In vitro, in addition to blocking IL-8 binding to human neutrophils, K4.3 and K2.2 blocked a number of IL-8-dependent cellular functions including neutrophil activation, up-regulation of the cell adhesion receptor CD11b/CD18, and neutrophil chemotaxis, suggesting that the fully human anti-IL-8 mAbs derived from XenoMouse strains are potent anti-inflammatory agents. This was further supported by in vivo studies in which K4.3 and K2.2 significantly inhibited IL-8-induced skin inflammation in rabbits. A pharmacokinetic study in Cynomolgus monkeys demonstrated that the alpha phase half-life is 9.4 h and the beta phase 10.9 days, typical of human mAbs in monkeys. These data support advancing a fully human anti-IL-8 mAb into clinical trials to treat inflammatory diseases.

Cytokine-mediated inflammatory hyperalgesia limited by interleukin-10.

1. The effect of interleukin-10 (IL-10) upon the hyperalgesic activities in rats of bradykinin, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), prostaglandin E2 (PGE2) and carrageenin were investigated in a model of mechanical hyperalgesia. 2. Hyperalgesic responses to bradykinin (1 micrograms) were inhibited in a dose-dependent manner by prior treatment with IL-10 (1-100 ng). 3. Hyperalgesic responses to TNF alpha (2.5 pg), IL-1 beta (0.5 pg) and IL-6 (1.0 ng) but not to IL-8 (0.1 ng) and PGE2 (50 ng and 100 ng) were inhibited by prior treatment with IL-10 (10 ng). 4. Hyperalgesic responses to carrageenin (100 micrograms) were inhibited by IL-10 (10 ng) when this cytokine was injected before but not after the carrageenin. 5. A monoclonal antibody to mouse IL-10 potentiated the hyperalgesic responses to carrageenin (10 micrograms) and TNF alpha (0.025 pg) but not that to IL-8 (0.01 ng). 6. In in vitro experiments in human peripheral blood mononuclear cells (MNCs), IL-10 (0.25-4.0 ng ml-1) inhibited in a dose-dependent manner PGE2 production by MNCs stimulated with IL-1 beta (1-64 ng ml-1) or endotoxin (lipopolysaccharide, LPS, 1 iu = 143 pg ml-1) but evoked only small increases in IL-1ra production. 7. These data suggest that IL-10 limits the inflammatory hyperalgesia evoked by carrageenin and bradykinin by two mechanisms: inhibition of cytokine production and inhibition of IL-1 beta evoked PGE2 production. Our data suggest that the latter effect is not mediated via IL-10 induced IL-Ira and may result from suppression by IL-10 of prostaglandin H synthase-2 (COX-2).

Cytotoxicity of cytokines in cerebral microvascular endothelial cell.

OBJECTIVE: Several studies reported that the levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells. METHOD: The effects of TNF-alpha, IL-1beta, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity. RESULTS: TNF-alpha and IL-1beta, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner (p<0.05). TNF-alpha (200 pg/ml) and IL-1beta (150 pg/ml) produced DNA ladders at 24-72 h. TNF-alpha but not IL-1beta cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24-72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 micromol/l significantly prevented TNF-alpha-induced cell detachment (p<0.05). DISCUSSION: TNF-alpha induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1beta decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1beta may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-alpha.

Cytokine-associated tissue injury and lethality in mice: a comparative study.

A comparative study was performed to examine the lethal effects of several cytokines injected into mice sensitized with actinomycin D (Act-D). Consistent with published data, human tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) (0.2-5 micrograms) caused the death of the animals within 8-12 hr after injection. Human interleukin-6 (IL-6) and interleukin-8 (IL-8) (0.6-6 micrograms) known to be induced by TNF-alpha did not show any lethal effects, indicating that TNF-alpha-associated lethality is not mediated by IL-6 or IL-8. Human tumor necrosis factor-beta (TNF-beta) (also called lymphotoxin), which shares structural and functional properties with TNF-alpha, was as potent as TNF-alpha in its lethal effects. Murine interferon-gamma (IFN-gamma) (0.04-5 micrograms) was also tested and showed no lethal effects in this model. In addition, a synthetic peptide corresponding to amino acid residues 163-171 of IL-1 beta, and which has been shown to lack the inflammatory effects of IL-1 beta, also caused no lethality among Act-D sensitized mice. The pretreatment of mice with IL-6, IL-8, or IFN-gamma had no protective effects on TNF-alpha or IL-1 beta-induced lethality in contrast to the protection observed by a pretreatment with TNF-alpha/IL-1 beta themselves or with endotoxin. Histopathologic data showed that severe tissue injury in vital organs is associated with the rapid lethality among sensitized mice.

Viruria during acute Japanese encephalitis virus infection.

In this study, viruria following Japanese encephalitis virus (JEV) infection in mice has been shown to appear earlier in pregnant than in normal mice with proteinuria and haematuria. This was related to the production of splenic macrophage derived neutrophil chemotactic factor (MDF) following JEV infection. Intravenous inoculation of MDF in mice resulted in leakage of cells, proteins and erythrocytes in the urine as a result of altered capillary permeability. The isolation of virus from kidney did not correlate with the shedding of virus in the urine. The histological examination of sections of kidneys showed no morphological damage; however, ultrastructural degenerative changes in the mesangial cells were observed following JEV infection. These data suggest that JEV-induced macrophage derived factor regulates the leakage of proteins, erythrocytes and cells into the urine.

Release of polymorphonuclear leukocytes from the bone marrow by interleukin-8.

Several studies have shown that interleukin-8 (IL-8) causes a rapid granulocytosis with the release of polymorphonuclear leukocytes (PMN) from the bone marrow (BM) partially responsible for the granulocytosis. This study was designed to quantitate the release of PMN from the BM by IL-8 and measure the transit time of PMN through the marrow after IL-8 administration. The thymidine analogue, 5'-bromo-2'-deoxyuridine (BrdU), was used to label dividing PMN in the marrow and follow their release into the circulation after intravenous IL-8. This allowed us to calculate the transit time of PMN through the mitotic and postmitotic pools of BM. BrdU was infused intravenously into rabbits 24 hours before IL-8 (2.5 microg/kg). IL-8 caused a rapid, transient granulocytopenia (5.9 +/- 0.4 at baseline v 0.2 +/- 0.06 x 10/9L at 5 minutes, P < .05) followed by granulocytosis (8.4 +/- 0.1 at 30 minutes, P < .05) associated with an increased number (0.3 +/- 0.1 at baseline v 1.2 +/- 0.6 x 10(9)/L at 30 minutes, P < .05) and percentage of band cells (P < .05), as well as a rapid increase in the number of BrdU-labeled PMN (PMNBrdU) in the circulation (0.09 +/- 0.05 at baseline to 1.5 +/- 0.6 x 10(9)/L at 60 minutes, P < .05). The transit time of PMN through both the mitotic and postmitotic pools of BM was not affected by IL-8. To determine the marrow compartment from which the PMN were mobilized by IL-8, we quantitated PMN movement from the hematopoietic and sinusoidal compartments into the circulation. The fraction of PMNBrdU in both compartments was higher than in the circulating blood (P < .05) and the fraction and number of PMNBrdU in the sinusoids decreased with IL-8 treatment (P < .05). We conclude that the pool of PMN residing in the BM venous sinusoids are rapidly released into the circulation after administration of IL-8.

In vitro and in vivo activity and pathophysiology of human interleukin-8 and related peptides.

Interleukin-8 is a member of a novel cytokine family and has been found to be an activator and attractant for human neutrophils in vitro. The in vivo activity was tested in experimental animal models by intradermal and intravenous administration of IL-8. Intradermal administration of human IL-8 in rats induces a rapid and concentration-dependent neutrophil infiltration, which peaks 4 hr after IL-8 application. Injection of GRO-alpha induces a similar chemotactic response, whereas neutrophil-activating peptide-2 was significantly less active. When injected intravenously into rabbits, IL-8 induced neutrophil sequestration in the lungs and, following repeated injections, caused septal and intraalveolar edema and lung damage resembling that seen in adult respiratory distress syndrome. The fact that IL-8 is induced and secreted from many different cell types suggests its involvement in a variety of physiologic and pathologic conditions as a neutrophil chemoattractant and, possibly, as an activator of other neutrophil responses.