TNF-alpha Priming Research Articles

TNF-Alpha Priming Enhances CD4+FoxP3+ Regulatory T Cell Suppressive Function in GVHD Prevention and Treatment

TNF-Alpha Priming Enhances CD4+FoxP3+ Regulatory T Cell Suppressive Function in GVHD Prevention and Treatment

TNF-Alpha Priming Enhances CD4+ FoxP3+ Regulatory T Cell Suppressive Function in GvHD Prevention and Treatment

CD4+ CD25+ FoxP3+ regulatory T cells (Treg) have been shown to effectively prevent graft versus host disease (GvHD) when adoptively transferred in murine models of hematopoietic cell transplantation (HCT) and phase I/II clinical trials. Critical limitations to the clinical application of Treg are the paucity of cells and limited knowledge of the mechanism(s) of in vivo function.In physiologic conditions Treg regulate immune responses during inflammation. We hypothesized that inflammatory conditions in GvHD modify Treg characteristics and function. To test this hypothesis, we primed Treg with irradiated (3000 cGy) peripheral blood from acute GvHD (aGvHD) affected mice for 20-24 hours and then transferred these cells in a mouse model of GvHD where allogeneic T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/c recipients together with 7.5x105 to 1x106 /animal donor derived conventional CD4+ and CD8+ T cells (Tcon). C57BL/6 Treg primed with irradiated aGvHD peripheral blood were injected at day 0 after HCT for preventing GvHD or at day +7 or +8 as GvHD treatment. Their adoptive transfer resulted in improved survival in comparison to unprimed natural occurring Treg when used for both GvHD prevention (p=0.01) and treatment (p=0.04). Moreover treatment with irradiated aGvHD peripheral blood-primed Treg did not impact graft versus tumor effects in a mouse model of T cell mediated tumor killing. BLI demonstrated that injected allogeneic Tcon completely cleared previously infused luc+ A20 tumor cells even in the presence of primed Treg (primed Treg + Tcon + A20 vs A20 alone p<0.001). Irradiated aGvHD peripheral blood-primed Treg express increased levels of activation markers with suppressive function such as CTLA4 (p<0.001) and LAG3 (p<0.05) in comparison to unprimed Treg in vitro. We also found that Treg primed with irradiated cells of aGvHD affected animals after removing the serum did not enhance the expression of the same markers (p>0.05) demonstrating that serum from aGvHD animals is required for Treg priming and function. We further tested the ability of several inflammatory cytokines that are normally secreted during GvHD such as IFN-γ, IL-6, IL-12 and TNFα to induce similar in vitro Treg activation. We found that TNFɑ selectively activated Treg without impacting CD4+ FoxP3- T cells. TNFɑ-primed Treg have increased expression of activation markers such as CD69 (p<0.0001), CD25 (p<0.0001), and LAG3 (p=0.0002), produce a greater amount of suppressive cytokines such as IL-10 (p=0.03) and TGF-β (p=0.02), and enhance the expression of homing markers such as CD62L (p=0.005) that are required for in vivo function. TNFɑ-primed Treg had increased ability to proliferate (p=0.02) and, at the same time, to suppress Tcon proliferation (p=0.04) in a mixed lymphocyte reaction against irradiated allogeneic splenocytes, while, on the contrary, TNFɑ-primed Tcon had reduced ability to proliferate in similar conditions in comparison to unprimed Tcon (p=0.0004). To test the effect of TNFɑ priming on in vivo Tcon proliferation we injected TNFɑ-primed and unprimed luc+ Tcon in allogeneic BALB/c Rag2-/- γ-chain-/- immune deficient animals that were sublethally irradiated (400 cGy). BLI at day +7 after Tcon injection revealed reduced TNFɑ-primed Tcon in vivo proliferation (p=0.01) that resulted in milder GvHD symptoms (p=0.02). Finally, in a GvHD prevention mouse model TNFɑ-primed Treg infused at 1:10 Treg/Tcon ratio resulted in improved animal survival as compared to unprimed Treg (p=0.02), demonstrating enhanced efficacy of TNFɑ priming in the in vivo function of Treg.In summary, our study demonstrates that Treg respond to TNFɑ acquiring an activated status resulting in increased function. As TNFɑ is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore TNFɑ priming of Treg in vitro provides a new tool to optimize Treg cellular therapies also allowing for the use of a reduced cell number for GvHD prevention and treatment. DisclosuresNo relevant conflicts of interest to declare.

The Cleavage of Neutrophil Leukosialin (CD43) by Cathepsin G Releases Its Extracellular Domain and Triggers Its Intramembrane Proteolysis by Presenilin/γ-Secretase

The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-alpha priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by alpha1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/gamma-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different gamma-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/gamma-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.

TNF-α priming effect on polymorphonuclear leukocyte reactive oxygen species generation and adhesion molecule expression in hemodialyzed patients

.IntroductionThe study aimed to assess reactive oxygen species generation and the expressions of some surface antigens on polymorphonuclear leukocytes (PMNs) in patients on regular hemodialysis (HD) treatment.Materials and MethodsThe respiratory burst of PMNs was determined with luminol-dependent chemiluminescence (CL) in resting cells and following N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), or opsonized zymosan (OZ) stimulation and expressed in arbitrary CL units times assay-time (aU × min). The expressions of CD11b/CD18, CD10, and CD13 receptors were determined with flow cytometry.ResultsBasal PMN CL was increased in HD patients to up to 1285 ± 129 aU × min compared with 895 ± 88 aU × min in healthy controls (p < 0.05). The CL of unprimed PMNs increased after fMLP stimulation from 3085 ± 746 to 4529 ± 808 aU × min, and after OZ stimulation from 12945 ± 1296 to 14678 ± 1355 aU × min. PMA-stimulated CL of PMNs was similar to control values. The oxidative burst in PMNs from HD patients and healthy controls was similar in response to TNF-α alone. The CL of TNF-α-primed PMNs in HD patients was significantly lower than CL measured in healthy controls (p < 0.05). The expressions of CD10 and CD13 metalloproteinase receptors were also increased (p < 0.05). Although CD11b expression was significantly increased at rest and after fMLP stimulation, the expression of another β-integrin heterodimer compound, CD18, was not increased.ConclusionsThese results provide evidence that TNF-α priming of PMNs is down-regulated in HD patients despite constitutive up-regulation of resting cytotoxicity and enhanced expression of adhesion and metalloproteinase receptors.

IL-18 is upregulated in the kidney and primes neutrophil responsiveness in ANCA-associated vasculitis

In antineutrophil cytoplasm autoantibody (ANCA)-associated systemic vasculitis (ASV), autoantibody-induced neutrophil activation is believed to cause organ damage. In vitro, tumor necrosis factor alpha (TNFalpha) primes neutrophils for ANCA stimulation and TNFalpha blockade has been successfully used to treat ASV. Nonetheless, irreversible organ damage can still occur, suggesting that other cytokines may circumvent TNFalpha blockade. We report that interleukin (IL)-18 deposition, as assessed by immunoperoxidase staining, is increased in renal biopsies from ASV patients. Immunofluorescence microscopy demonstrated that podocytes are the predominant glomerular IL-18-positive cell type, whereas in the interstitium, myofibroblasts, distal tubular epithelium, and infiltrating macrophages stained for IL-18. In vitro, IL-18 primed superoxide production by ANCA-activated neutrophils comparably to TNFalpha. IL-18-primed, ANCA-induced superoxide production was unaffected by anti-TNFalpha antibody, which abrogated TNFalpha priming. Furthermore, TNFalpha and IL-18 phosphorylated neutrophil p38 mitogen-activated protein kinase (MAPK), but IL-18-mediated p38 MAPK phosphorylation was unaffected by anti-TNFalpha antibody. The p38 MAPK inhibitor, SB20358, reduced IL-18-primed, ANCA-induced superoxide production in a concentration-dependent manner. ANCA-induced superoxide release was also sensitive to the Leukotriene B4 (LTB4) inhibitor MK-886. IL-18 priming was not associated with increased ANCA antigen expression on isolated neutrophils. We conclude that IL-18 is likely to be important for neutrophil recruitment and priming in ASV. Therapies targeting single priming agents may have limited efficacy in controlling disease.

Effects of Arsenic on Zebrafish Innate Immune System

The innate immune response, the first line of defense against invading pathogens, can be perturbed by environmental toxicants such as arsenic. This study reports the effects of arsenic on innate immunity of zebrafish. Respiratory burst activity, messenger RNA expression of tumor necrosis factor alpha (TNF-alpha), a primer of the respiratory burst response, and mRNA expression of the antiviral cytokines interferon (IFN) and MX, : before and after viral infection, were examined in arsenic-exposed zebrafish larvae. Respiratory burst activity and TNF-alpha expression were decreased upon arsenic exposure, indicating inhibition of TNF-alpha priming of the respiratory burst response. Arsenic enhanced IFN expression slightly over time, but reduced MX : expression. In zebrafish infected with snakehead rhabdovirus, arsenic decreased induction and altered the kinetics of IFN and MX : upon infection. Differences in IFN and MX : expression in arsenic-exposed larvae point toward an interruption of the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway.

Distinct Ligand-dependent Roles for p38 MAPK in Priming and Activation of the Neutrophil NADPH Oxidase

In response to certain cytokines and inflammatory mediators, the activity of the neutrophil NADPH oxidase enzyme is primed for enhanced superoxide production when the cells receive a subsequent oxidase-activating stimulus. The relative role of p38 MAPK in the priming and activation processes is incompletely understood. We have developed a 2-step assay that allows the relative contributions of p38 MAPK activity in priming to be distinguished from those involved in oxidase activation. Using this assay, together with in vitro kinase assays and immunochemical studies, we report that p38 MAPK plays a critical role in TNFalpha priming of the human and porcine NADPH oxidase for superoxide production in response to complement-opsonized zymosan (OpZ), but little, if any, role in neutrophil priming by platelet-activating factor (PAF) for OpZ-dependent responses. The OpZ-mediated activation process per se is independent of p38 MAPK activity, in contrast to oxidase activation by fMLP, where 70% of the response is eliminated by p38 MAPK inhibitors regardless of the priming agent. We further report that incubation of neutrophils with TNFalpha results in the p38 MAPK-dependent phosphorylation of a subpopulation of p47(phox) and p67(phox) molecules, whereas PAF priming results in phosphorylation only of p67(phox). Despite these phosphorylations, TNFalpha priming does not result in significant association of either of these oxidase subunits with neutrophil membranes, demonstrating that the molecular basis for priming does not appear to involve preassembly of the NADPH oxidase holoenzyme/cytochrome complex prior to oxidase activation.

Particle-epithelial interaction: effect of priming and bystander neutrophils on interleukin-8 release.

Exposure to ambient air pollution particles causes greater health effects in individuals with preexisting inflammatory lung diseases. To model inflammatory priming in vitro, HTB54 lung epithelial cells were pretreated with tumor necrosis factor-alpha (TNF-alpha) and then exposed to a panel of environmental particles, including concentrated ambient particles (CAPs). TNF-alpha priming significantly enhanced interleukin (IL)-8 secretion in response to CAPs and other urban air particles in HTB54 cells. Enhancement was seen with whole CAP suspensions as well as with its separate water-soluble and -insoluble components. Treating CAP suspensions with 20 microM deferoxamine or 2 mM dimethylthiourea attenuated the enhancement, indicating that transition metals and oxidative stress participate in the CAPs-dependent IL-8 response of primed cells. Because activated neutrophils are also present in diseased lungs and are sources of additional oxidative stress on epithelial cells, primed HTB54 cells were cocultured with activated neutrophils. Wild-type neutrophils markedly enhanced IL-8 release to CAPs in primed HTB54 cells, an effect substantially diminished when neutrophils from NADPH knockout mice were used. Cytokine priming and interactions with activated neutrophils can amplify lung epithelial inflammatory responses to ambient air particles.

Extracellular signal-regulated kinase inhibition by statins inhibits neutrophil activation by ANCA

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may modulate cellular inflammatory functions independent of serum cholesterol. We tested the hypothesis that statins decrease respiratory burst activity of human polymorphonuclear neutrophils (PMN) in response to anti-neutrophil cytoplasmic antibodies (ANCA). Neutrophils were isolated from healthy human volunteers, human immunoglobulins were isolated from patients with proteinase-3 (PR3)- and myeloperoxidase (MPO)-ANCA. Superoxide generation was measured by the ferricytochrome C assay and the nitro blue tetrazolium (NBT) test. ANCA antigen expression was measured by flow cytometry and phosphorylation of mitogen-activated protein kinase (MAPK) was assessed by Western blotting. Cerivastatin and simvastatin inhibited respiratory burst activity to ANCA dose-dependently (1 to 25 micromol/L). Tumor necrosis factor-alpha (TNF-alpha)-primed neutrophils released 26.7 +/- 2.8 nmol O2-/0.75 x 106 PMN/45 min and 10 micromol/L simvastatin reduced this amount to 18.0 +/- 2.1 nmol. The inhibitory effect was confirmed by the NBT test. The respiratory burst decrease could not be reversed by 500 micromol/L mevalonic acid (MVA). In this assay, both statins also inhibited the response to human ANCA. PR3-ANCA resulted in 19.4 +/- 2.0 nmol O2- nmol. This amount was decreased to 6.0 +/- 1.2 nmol by preincubation with 10 micromol/L simvastatin (P < 0.01). For MPO-ANCA, the values were 22.6 +/- 2.8 nmol for controls versus 16.7 +/- 3.1 nmol with statin (P < 0.01). By FACS, simvastatin decreased TNF-alpha-mediated ANCA antigen translocation (from 219 +/- 33 to 180 +/- 35 MFI for PR3 and 24.0 +/- 2.4 to 18.3 +/- 1.1 for MPO). Finally, since p38 MAPK and ERK control TNF-alpha priming, we studied the effects of both statins on MAPK. Western blotting showed that statins inhibited TNF-alpha-induced ERK phosphorylation in a dose dependent fashion, but had no effect on p38. These findings demonstrate that HMG-CoA reductase inhibitors decrease respiratory burst activity of human PMN in response to ANCA. This effect was independent of mevalonate, but involved inhibition of ERK activation during TNF-alpha priming. Our data suggest that HMG-CoA reductase inhibitors may help limit inflammatory responses.

Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils.

Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor alpha (TNF-alpha) in vitro. Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-alpha-primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3- and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-alpha and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-alpha priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation. A total of 20 +/- 3 nmol O(2)(-)/0.75 x 10(6) PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 microM) decreased this amount to 0.3 +/- 2.6 nmol (n = 10, P < 0.05); the MPO-ANCA values were 23 +/- 3 versus 1.6 +/- 3.6 (n = 10, P < 0.05). p38 MAPK inhibition with 10 microM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-alpha and to ANCA. However, SB202190 but not LY294002 abrogated TNF-alpha-mediated ANCA antigen surface translocation, demonstrating that superoxide generation and ANCA antigen translocation proceed by separate mechanisms. Akt, PAK1, and Rac1 existed as cytosolic complex in resting PMN. TNF-alpha stimulation increased association of PAK1 with Akt. An MPO monoclonal antibody did not alter the Akt signaling complex further. The data demonstrate the importance of PI3-K for the ANCA-induced PMN oxidant production.

Role of mitogen-activated protein kinases in activation of human neutrophils by antineutrophil cytoplasmic antibodies.

Antineutrophil cytoplasmic antibodies (ANCA) may be important in the pathophysiology of necrotizing vasculitis. ANCA activate human neutrophils primed with tumor necrosis factor-alpha (TNF-alpha) in vitro. TNF-alpha priming results in translocation of ANCA antigens to the cell surface, where they are recognized by the antibodies. The signaling mechanisms involved in TNF-alpha priming and subsequent ANCA-induced activation were investigated. TNF-alpha-primed neutrophils were stimulated with monoclonal antibodies (MAb) to human myeloperoxidase (MPO) and proteinase 3 (PR3), and with preparations of human ANCA (three patients with PR3-ANCA and two patients with MPO-ANCA). Respiratory burst was measured with superoxide dismutase-inhibitable ferricytochrome C reduction and using dihydro-rhodamine-1,2,3. Phosphorylation of p38 mitogen-activated protein kinase (p38-MAPK) and the extracellular signal-regulated kinase (ERK) were assessed by immunoblotting. ANCA-antigen translocation was studied by flow cytometry. The tyrosine phosphorylation inhibitor genistein, but not calphostin or staurosporin, resulted in a significant dose-dependent superoxide generation inhibition (11.6 +/- 1.7 nmol to 2.1 +/- 0.5 for PR3-ANCA, and 16.0 +/- 2.8 to 3.3 +/- 1.3 for MPO-ANCA). The p38-MAPK inhibitor (SB202190) and the ERK inhibitor (PD98059) diminished PR3-ANCA-mediated superoxide production dose dependently (11.6 +/- 1.7 nmol O(2)(-) to 1.9 +/- 0.6 with 50 microM SB202190 and 4.0 +/- 0.6 with 50 microM PD098059, respectively). For MPO-ANCA, the results were similar (16.0 +/- 2.8 nmol to 0.9 +/- 1.0 nmol with SB202190 and 6.4 +/- 2.4 nmol with PD98059, respectively). Western blot showed phosphorylation of both p38-MAPK and ERK during TNF-alpha priming. The p38-MAPK inhibitor and the ERK inhibitor showed the strongest effect on respiratory burst when added before TNF-alpha priming, further supporting an important role for both signaling pathways in the priming process. Flow cytometry showed that p38-MAPK inhibition decreased the translocation of PR3 (by 93 +/- 2%) and of MPO (by 64 +/- 2%). In contrast, no such effect was seen when ERK was inhibited. Thus, p38-MAPK and ERK are important for the TNF-alpha-mediated priming of neutrophils enabling subsequent ANCA-induced respiratory burst. However, both pathways show differential effects, whereby p38-MAPK controls the translocation of ANCA antigens to the cell surface.

IgG from myeloperoxidase-antineutrophil cytoplasmic antibody-positive patients stimulates greater activation of primed neutrophils than IgG from proteinase 3-antineutrophil cytoplasmic antibody-positive patients

Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) have been reported to be pathologically and clinically different. The aim of this study was to assess whether these differences could be explained by differing abilities of proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA)-positive IgG preparations or myeloperoxidase-ANCA (MPO-ANCA)-positive IgG preparations to activate neutrophils (polymorphonuclear cells [PMN]) in vitro. Using Percoll density gradients, PMN were isolated (concentration 2 x 10(6)/ml) and primed with cytochalasin B (1 ng/ml) and tumor necrosis factor alpha (TNFalpha; 2 ng/ml). The PMN were activated with 200 microg/ml of normal IgG or ANCA. Activation was determined by 1) superoxide anion generation as determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c, 2) monitoring fluxes in Ca2+ concentration using Fura 2-AM-loaded PMN, and 3) degranulation using an MPO assay. Surface expression of PR3 and MPO was determined by fluorescence-activated cell sorter analysis. ANCA isotypes were investigated by enzyme-linked immunosorbent assay. Activation of PMN by MPO-ANCA-positive IgG preparations compared with PR3-ANCA-positive IgG preparations resulted in greater generation of superoxide anions (MPO-ANCA-positive IgG preparations 9.13 +/- 0.39 nmoles [mean +/- SEM], PR3-ANCA-positive IgG preparations 6.32 +/- 0.35 nmoles; P < 0.001), Ca2+ fluxes (MPO-ANCA-positive IgG preparations 0.735 +/- 0.10, PR3-ANCA-positive IgG preparations 0.33 +/- 0.098; P < 0.01), and MPO degranulation (MPO-ANCA-positive IgG preparations 251.98 +/- 26.7 ng, PR3-ANCA-positive IgG preparations 145.19 +/- 19.4 ng; P < 0.001). The increased activation seen with MPO-ANCA-positive IgG preparations was not due to increased expression of MPO on the cell surface, because following TNFalpha priming PR3 was expressed on significantly more cells than was MPO (PR3 expression 54.2 +/- 5.18%, MPO 31.6 +/- 3.55%; P < 0.001). IgG1 and IgG4 were the predominant isotypes in both MPO-ANCA-positive IgG preparations and PR3-ANCA. MPO-ANCA contained significantly more IgG1 than did PR3-ANCA, and PR3-ANCA-positive IgG preparations contained significantly more IgG3. In vitro MPO-ANCA-positive IgG preparations are more activating than PR3-ANCA-positive IgG preparations. The increased activation cannot be explained by increased MPO expression on the cell surface or greater IgG3 present in MPO-ANCA-positive IgG preparations. Differences in activation of PMN by these antibodies may determine some differences between WG and MPA.

Diesel exhaust particles induced release of interleukin 6 and 8 by (primed) human bronchial epithelial cells (BEAS 2b) in vitro

Several epidemiological studies have recently shown associations of increased premature mortality rates with ambient particulate air pollution. Diesel exhaust particles (DEP) may constitute an important part of (ultra)fine particulate air pollution in urban areas and may therefore contribute to its toxicity. Epithelial lining of the respiratory tract may be the first target of the toxic effects of DEP, that upon exposure may release pro-inflammatory mediators such as interleukin 6 and 8 (IL-6, IL-8), ultimately causing airway tissue damage and immune alterations. In this study the effects of in vitro DEP exposure (0.04-0.33 mg/mL) on IL-6, IL-8 production by a human bronchial epithelial cell line (BEAS-2B) were investigated. For comparison, the production of interleukins during exposure to silica and titanium oxide (TiO2) were also studied, representing relatively toxic and non-toxic particles, respectively. Scanning and transmission electron microscopy showed that the size of the DEP particles ranged between 25 to 35 nm and that DEP was phagocytized by BEAS-2B cells. An increase in IL-6 and IL-8 production (11- and 4-fold, respectively) was found after 24 or 48 h of exposure to DEP compared to the non-exposed cells. This increase was lower compared to silica (17- and 3.3-fold) and higher as compared to TiO2 which showed no increase for IL-6 and IL-8. To study the DEP effect on inflammation-primed cells, BEAS-2B cells were exposed to both tumor necrosis factor-alpha (TNF-alpha) and subsequently to DEP. Exposure to TNF-alpha caused a strong increase in IL-6 and IL-8 production. Additive effects on the IL-6 and IL-8 production by BEAS-2B cells were found after TNF-alpha priming and subsequently exposure to DEP, only at a low dose of DEP and TNF-alpha (0.05-0.2 ng/mL). In conclusion, BEAS-2B phagocytized DEP and produced an increased amount of IL-6 and IL-8. In TNF-alpha primed BEAS-2B cells, DEP increased interleukin production only at low concentrations of DEP and TNF-alpha. Whether this increased production of pro-inflammatory interleukins affects vulnerable balances in the immune system, such as T help-1 and T help-2 subsets ratios, resulting in an altered resistance to respiratory tract infections or altering the expression of respiratory allergy, is the subject of further studies.

The loss of recombinant human granulocyte colony-stimulating factor and recombinant human TNF-alpha priming effects on the superoxide-generating response in exudated neutrophils is associated with a decrease in their receptor affinities.

Several cytokines are known to enhance FMLP-stimulated superoxide generation in human circulating blood neutrophils through binding to their specific receptors, a process referred to as the priming effect. The priming effects produced by recombinant human granulocyte CSF (rhGCSF) and TNF-alpha (rhTNF-alpha) on FMLP-stimulated superoxide production in human and rabbit blood neutrophils were compared with their effects in their respective tissue neutrophils, i.e., human salivary and rabbit peritoneal neutrophils. The receptor binding characteristics of rhGCSF and rhTNF-alpha were also compared between the two types of neutrophils. Both rhGCSF and rhTNF-alpha produced dose-dependent priming effects on FMLP-stimulated superoxide production in human blood neutrophils, whereas they failed to produce any priming effects in human salivary neutrophils. Similar results were obtained for the priming effects by rhGCSF in rabbit blood and peritoneal neutrophils. A decrease in receptor binding affinity, but not in receptor density, in tissue neutrophils was demonstrated by analyzing the binding of [125I]rhGCSF and [125I]rhTNF-alpha. These findings suggest that tissue neutrophils are less responsive to rhGCSF and rhTNF-alpha in the modulation of FMLP-stimulated superoxide generation. This is due at least in part to the lower affinities of GCSF and TNF-alpha to their receptors in tissue neutrophils. This marked difference in priming effects by cytokines between blood and tissue neutrophils may represent an early step in the defensive responses against invading microorganisms or Ags.

Molecular and functional evidence for in vitro cytokine enhancement of human and murine target cell sensitivity to glucocorticoids. TNF-alpha priming increases glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis.

Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-alpha increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-alpha also enhanced GR number. The TNF-alpha effect on transcriptional activity was absent in other cell lines that express TNF-alpha receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-alpha, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-alpha increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L-929 cells with a low (noncytotoxic) dose of TNF-alpha significantly increased the sensitivity to glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis. TNF-alpha and IL-1 beta had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Tumor necrosis factor-α augments the pro-inflammatory interaction between PMN and GBM via a CD 18 dependent mechanism

Acute glomerulonephritis is frequently associated with intraglomerular neutrophil (PMN) accumulation and the intensity of the inflammatory reaction is correlated with elevated concentrations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF alpha). PMN are thought to damage glomeruli due to a combination of reactive oxygen species and proteolytic enzymes. Using an in vitro model of anti-GBM nephritis the effects of TNF alpha on GBM damage by PMN were evaluated. The interaction of GBM and PMN resulted in a low grade respiratory burst that was significantly augmented by the addition of TNF alpha. Luminol dependent chemiluminescence (LCL) was increased from 2.4 x 10(6) to 48.1 x 10(6) (P < 0.05). The GBM induced LCL could be > 85% inhibited by blocking with monoclonal antibodies (mAbs) to the common beta chain of the PMN beta 2 integrin family (CD18), but was unaffected by mAbs to CD11a or CD11b subunits. Degradation of GBM, however, was not influenced by either TNF alpha priming of PMN or anti-beta 2 integrin mAbs. When PMN were incubated with GBM-anti-GBM IgG complex they underwent an increase in LCL from 2.4 x 10(6) to 31.1 x 10(6). They also degraded more GBM than controls (10.1% vs. 1.8%). These aspects of PMN activation were Fc receptor mediated, dependent upon anti-GBM IgG being bound to GBM and inhibited by mAb to the PMN Fc receptor. These studies show that TNF alpha can modulate the inflammatory response of PMN in contact with GBM in a CD18 dependent manner. In contrast, Fc receptor mediated events are uninfluenced by TNF alpha.

Dissociation of TNF-alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.

Human tumour necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF-alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF-alpha can be dissociated. The TNFR55-selective mutants (R32W, E146K and R32W-S86T) which bind poorly to TNFR75 displayed similar potency to wild-type TNF in causing cytotoxicity of a human laryngeal carcinoma-derived cell line (HEp-2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55-selective mutants exhibited lower proinflammatory activity than wild-type TNF. Specifically, TNF-alpha's priming of human neutrophils for superoxide production and antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis and adhesion to endothelium were reduced by up to 170-fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E-selectin expression, neutrophil transmigration and IL-8 secretion were also reduced by up to 280-fold. On the other hand, D143F, a TNFR75-selective mutant tested either alone or in combination with TNFR55-selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75-transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant tumor necrosis factor-alpha potentiates neutrophil degranulation in response to host defense cytokines neutrophil-activating peptide 2 and IL-8 by modulating intracellular cyclic AMP levels.

The neutrophil-activating peptide 2 (NAP-2) and IL-8 are closely related in structure and function. In order to further determine their potential biologic roles in inflammation, we studied their interaction with TNF-alpha-primed human polymorphonuclear neutrophil granulocytes at the levels of effector functions and signal transduction. After short term priming (5 min) by TNF-alpha, suspended cytochalasin B-treated PMN responded to NAP-2 or rIL-8 by substantial augmentation of the degranulation response. After priming with 3 ng/ml TNF-alpha marker release from both azurophilic and specific granules was near maximum. NAP-2 and rIL-8 cooperated with TNF-alpha in very similar ways, as indicated by the almost identical increases in release rates that were induced by equipotent doses of either secondary stimulus. At the signal transduction level, pharmacologic elevation of intracellular cAMP led to the inhibition of NAP-2- or rIL-8-induced degranulation in primed and unprimed PMN, indicating a role for this second messenger as a negative feedback signal. Direct measurement of intracellular cAMP revealed that TNF-alpha by itself did not affect its levels. Instead, TNF-alpha reduced both the scale as well as the duration of the cAMP burst generated in response to secondary stimuli NAP-2 or rIL-8. Thus, there is evidence that TNF-alpha priming of neutrophils for enhanced NAP-2- or rIL-8-promoted degranulation involves the antagonistic down-modulation of stimulus-induced rises in cAMP.

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.

Tumor necrosis factor enhances the neutrophil‐dependent increase in endothelial permeability

We examined the effect of tumor necrosis factor alpha (TNF alpha) on the increase in pulmonary microvascular endothelial monolayer permeability induced by activated neutrophils (PMN). Layering of PMN onto endothelial monolayers followed by activation of PMN with phorbol 12-myristate 13-acetate (PMA) increased 125I-albumin clearance rate across the monolayers. Pretreatment of endothelial monolayers for 6 hr with TNF alpha (200 U/ml) potentiated the PMN-dependent increase in endothelial permeability, whereas 1 hr or 6 hr pretreatment of endothelial monolayers with 200 U/ml and 100 U/ml, respectively, TNF alpha did not enhance the response. Adherence of PMN to the endothelial cells was increased at 1 and 6 hr after TNF alpha (200 U/ml) treatment, but the adherence response was markedly greater following 6 hr of TNF alpha. The TNF alpha treatment of endothelial cells did not enhance neutrophil activation responses to PMA. Pretreatment of PMN with IB4, a MAb to the CD18 integrin, the common beta subunit of the adhesion proteins LFA-1, Mac-1, and p150,95 of PMN, reduced the increases in PMN adherence and the endothelial monolayer permeability induced by the 6 hr TNF alpha treatment. In contrast, pretreatment of PMN with OKM-1, a MAb to the CD11b epitope (alpha-subunit), had no effect on the adherence and the potentiation of the increase in permeability. The potentiation of the PMN-dependent permeability increase and enhanced endothelial adhesivity at 6 hr after TNF alpha priming of endothelial cells was dependent on protein synthesis. The results indicate that protein synthesis-dependent expression of an endothelial ligand for CD18 and resultant endothelial hyperadhesiveness potentiates the PMN-mediated increase in endothelial permeability after TNF alpha activation of endothelial cells. The priming of endothelial cells by TNF alpha may be a critical step in the mediation of endothelial injury.