Biomedical subjects

J K Kolls

Publications and source records attributed to J K Kolls.

At least 19 recordsLinked to original sources

PubMed2005

Joint inflammation and chondrocyte death become independent of Fcgamma receptor type III by local overexpression of interferon-gamma during immune complex-mediated arthritis.

OBJECTIVE: It has previously been shown that the onset and the degree of joint inflammation during immune complex (IC)-mediated arthritis depend on Fcgamma receptor type III (FcgammaRIII). Local adenoviral overexpression of interferon-gamma (IFNgamma) in the knee joint prior to onset of IC-mediated arthritis aggravated severe cartilage destruction. In FcgammaRI(-/-) mice, however, chondrocyte death was not enhanced by IFNgamma, whereas matrix metalloproteinase (MMP)-mediated aggrecan breakdown was markedly elevated, suggesting a role for the activating FcgammaRIII in the latter process. We undertook this study to determine the role of FcgammaRIII in joint inflammation and severe cartilage destruction in IFNgamma-stimulated IC-mediated arthritis, using FcgammaRIII(-/-) mice. METHODS: FcgammaRIII(-/-) and wild-type (WT) mice were injected in the knee joint with recombinant adenovirus encoding murine IFNgamma (AdIFNgamma) or with adenovirus encoding enhanced green fluorescent protein 1 day prior to induction of IC-mediated arthritis. Histologic sections were obtained 3 days after arthritis onset to study inflammation and cartilage damage. MMP-mediated expression of the VDIPEN neoepitope was detected by immunolocalization. Chemokine and FcgammaR expression levels were determined in synovial washouts and synovium, respectively. RESULTS: Injection of AdIFNgamma in naive knee joints markedly increased levels of messenger RNA for FcgammaRI, FcgammaRII, and FcgammaRIII. Upon IFNgamma overexpression prior to induction of IC-mediated arthritis, joint inflammation was similar in FcgammaRIII(-/-) and WT mice. The percentage of macrophages in the knee joint was increased, which correlated with high concentrations of the macrophage attractant macrophage inflammatory protein 1alpha. Furthermore, IFNgamma induced 2-fold and 3-fold increases in chondrocyte death in WT controls and FcgammaRIII(-/-) mice, respectively. Notably, VDIPEN expression also remained high in FcgammaRIII(-/-) mice. CONCLUSION: IFNgamma bypasses the dependence on FcgammaRIII in the development of IC-mediated arthritis. Furthermore, both FcgammaRI and FcgammaRIII can mediate MMP-dependent cartilage matrix destruction.

Aggrecans↗

PubMed2002

Retrovirus molecular conjugates.

Retrovirus-derived vectors are currently the preferred vectors used for human gene therapy protocols. Serious safety concerns persist, however, which are specifically related to the formation of a replication-competent virus, and no synthesis method currently employed precludes its formation with certainty. For many cell types, a low transduction efficiency results in insufficient therapeutic benefit. We describe the development of a molecular conjugate system, which permits transient chemical modification of a retrovirus with polylysine. This modification not only introduces additional safety features over standard unmodified retrovirus vectors, but also provides enhanced transduction efficiency.

Animals↗

PubMed2002

Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.

Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.

Animals↗

PubMed2001

Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice.

Many mutant mice deficient in leukocyte adhesion molecules display altered hematopoiesis and neutrophilia. This study investigated whether peripheral blood neutrophil concentrations in these mice are elevated as a result of accumulation of neutrophils in the circulation or altered hematopoiesis mediated by a disrupted regulatory feedback loop. Chimeric mice were generated by transplanting various ratios of CD18(+/+) and CD18(-/-) unfractionated bone marrow cells into lethally irradiated wild-type mice, resulting in approximately 0%, 10%, 50%, 90%, or 100% CD18 null neutrophils in the blood. The presence of only 10% CD18(+/+) neutrophils was sufficient to prevent the severe neutrophilia seen in mice reconstituted with CD18(-/-) bone marrow cells. These data show that the neutrophilia in CD18(-/-) mice is not caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. In CD18(-/-), CD18(-/-)E(-/-), CD18(-/-)P(-/-), EP(-/-), and EPI(-/-) mice, levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-17 (IL-17) were elevated in proportion to the neutrophilia seen in these mice, regardless of the underlying mutation. Antibiotic treatment or the propensity to develop skin lesions did not correlate with neutrophil counts. Blocking IL-17 or G-CSF function in vivo significantly reduced neutrophil counts in severely neutrophilic mice by approximately 50% (P <.05) or 70% (P <.01), respectively. These data show that peripheral blood neutrophil numbers are regulated by a feedback loop involving G-CSF and IL-17 and that this feedback loop is disrupted when neutrophils cannot migrate into peripheral tissues.

Animals↗

PubMed2001

Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Animals↗

PubMed2001

Il-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice.

Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin(-/low)c-kit(+)Sca1(+)). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45-75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.

Animals↗

PubMed2001

IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis.

T cell IL-17 displays proinflammatory properties and is expressed in the synovium of patients with rheumatoid arthritis. Its contribution to the arthritic process has not been identified. Here, we show that blocking of endogenous IL-17 in the autoimmune collagen-induced arthritis model results in suppression of arthritis. Also, joint damage was significantly reduced. In contrast, overexpression of IL-17 enhanced collagen arthritis. Moreover, adenoviral IL-17 injected in the knee joint of type II collagen-immunized mice accelerated the onset and aggravated the synovial inflammation at the site. Radiographic and histologic analysis showed markedly increased joint destruction. Elevated levels of IL-1beta protein were found in synovial tissue. Intriguingly, blocking of IL-1alphabeta with neutralizing Abs had no effect on the IL-17-induced inflammation and joint damage in the knee joint, implying an IL-1 independent pathway. This direct potency of IL-17 was underscored in the unabated IL-17-induced exaggeration of bacterial cell wall-induced arthritis in IL-1beta(-/-) mice. In conclusion, this data shows that IL-17 contributes to joint destruction and identifies an IL-1-independent role of IL-17. These findings suggest IL-17 to be a novel target for the treatment of destructive arthritis and may have implications for tissue destruction in other autoimmune diseases.

Adenoviridae↗

PubMed2001

Retrovirus molecular conjugates. A novel, high transduction efficiency, potentially safety-improved, gene transfer system.

Two significant barriers limit the use of amphotropic retrovirus for human gene transfer protocols: 1) low transduction efficiency in cells with low receptor expression and 2) safety concerns originating from the risk of formation and propagation of replication competent virus in vivo. In principle, if ecotropic retrovirus, which is incapable of infecting human cells, could be transiently modified to effectively transduce human cells, this safety risk could be alleviated. Here we demonstrate that formation of amphotropic retrovirus polylysine molecular conjugates (aMMLV-PL) enhanced gene transfer up to 10-fold in a variety of human cell lines over the equivalent of unconjugated vector (aMMLV). The polylysine modification and formation of ecotropic retrovirus molecular conjugates (eMMLV-PL) permitted effective and stable transduction of different human cell lines as well as primary human bone marrow stroma cells at frequencies of greater than 80%. It is conceivable that this novel ecotropic-based conjugate retrovirus vector could also potentially provide enhanced safety characteristics not only over amphotropic retrovirus vectors but also over genetically tropism-modified recombinant ecotropic vectors. In contrast to genetic modifications, physical or chemical modifications are not propagated. Thus, formation of replication competent eMMLV from conjugates would be self-limited and would not result in virus propagation in humans.

3T3 Cells↗

PubMed2001

Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in l6 myocytes and Fao hepatoma cells.

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.

Adenoviridae↗

PubMed2001

The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin.

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 microg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 microg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.

Animals↗

PubMed2001

Effects of adenovirus-mediated liver-selective overexpression of protein tyrosine phosphatase-1b on insulin sensitivity in vivo.

AIM: Protein tyrosine phosphatase-1B (PTP-1B) is an intracellular PTP known to dephosphorylate and inactivate upstream tyrosine phosphoproteins in the insulin signalling cascade. We and others reported increased abundance of catalytically impaired PTP-1B in tissue lysates from obese human subjects with and without type 2 diabetes, while genetic knockout of PTP-1B improves insulin sensitivity and prevents nutritionally mediated insulin resistance and obesity. The aim of the present work was to further elucidate the role of PTP-1B in glucose metabolism in vivo. METHODS: We used adenoviral constructs incorporating cDNAs for either wild-type (W/T) or a catalytically inactive C(215)S (C/S) mutant PTP-1B to achieve liver-selective PTP-1B overexpression in young Sprague-Dawley rats using tail vein injection, based on the high degree of hepatotropism of adenovirus 5 (Ad5). An Ad5-lacZ construct encoding beta-galactosidase was used as a control for viral effects alone. A hyperinsulinaemic euglycaemic clamp was used to study whole body glucose disposal and endogenous glucose production rates. RESULTS: Control studies in HIRcB cells confirmed catalytic activity and inactivity of W/T and C/S respectively. Mean PTP-1B abundance was 2.24 +/- 0.02- and 2.33 +/- 0.04-fold of saline-treated control in liver lysates of W/T and C/S rats respectively. Liver selective overexpression was confirmed by analysis of tissue lysates from liver, fat and muscle tissues. Ad5 treatment did not result in a statistically or clinically significant liver injury, as determined by serum alanine aminotransferase and histological examination. Seven days post injection, no significant difference in rate of weight gain, fasting blood glucose or insulin levels were seen in any group. Similarly, under steady-state glucose clamp conditions, glucose disposal rate (R(d)), endogenous glucose production rate (EGP) and serum insulin levels were similar in all groups. CONCLUSION: We conclude that moderate medium-term overabundance, to a degree resembling that seen in insulin-resistant states, of PTP-1B in liver tissue does not alter insulin action on glucose metabolism and that the major site of action of PTP-1B is presumably at insulin-responsive target tissue or tissues other than the liver.

Adenoviridae↗

PubMed2001

Gene therapy to modify pulmonary host defenses.

Respiratory infections remain a significant public health problem and are presently the 6th leading cause of death in the United States. Antibiotic-resistant organisms are encountered increasingly in both community-acquired and nosocomial infections. Despite progress in antibiotic development, biological-response modifiers may have increasing application to augment pulmonary host defenses against either drug-resistant infections or in high-risk hosts. Toward this end, gene therapy proposes to deliver biologicals as nucleic acids rather than protein. Gene therapy has the potential advantage of targeting the biological to specific cells or tissue compartments, and a more favorable pharmacokinetic profile. Data on gene delivery and efficacy in preclinical models of pulmonary infection are presented and discussed.

Animals↗

PubMed2001

Poly-L-lysine-based molecular conjugate vectors: a high efficiency gene transfer system for human progenitor and leukemia cells.

BACKGROUND: Targeted, specific receptor mediated gene transfer is a major goal of gene therapy research to accomplish gene transfer exclusively to the desired cell population. METHODS: First, the use of natural receptor for stem cell factor and transferrin receptor-targeted gene transfer using poly-L-lysine-based molecular conjugate vectors was evaluated in a panel of hematopoietic progenitor cell lines. Second, the ability of poly-L-lysine to enhance adenovirus mediated gene transfer efficiency was examined in different cell lines by using recombinant adenovirus-poly-L-lysine molecular conjugate conglomerates (recMCVEGFP). RESULTS: Despite effective ligand internalization receptor, gene expression amplification in receptor positive cell lines was not uniformly observed. Therefore, using a poly-L-lysine-based, receptor-targeted vector, neither transferrin nor natural receptor for stem cell factor mediated gene transfer can be considered a universally applicable procedure that exclusively depends on the presence of receptors on the cell surface; rather, it is a cell specific phenomenon. In our model, poly-L-lysine is the major contributor for gene transfer to hematopoietic progenitor cells, mediating the initial vector-cell binding. Human progenitor cell lines are poorly transduceable with recombinant adenovirus vectors. This new poly-L-lysine-modified, adenovirus-based vector could overcome virus tropism restrictions and consistently achieve very high transduction efficiency (>90%) in cells otherwise refractory to adenovirus gene transfer. CONCLUSIONS: Polylysine-based adenovirus vectors may have promise for situations in which high-efficiency gene transfer with transient high level transgene expression in hematopoietic cells is needed, such as leukemia vaccine protocols or for purging strategies in leukemia cell contaminated stem cell preparations.

Adenoviruses, Human↗

PubMed2001

Efficient c-kit receptor-targeted gene transfer to primary human CD34-selected hematopoietic stem cells.

We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.

Adenoviridae↗

PubMed2001

Interleukin-17 and lung host defense against Klebsiella pneumoniae infection.

Bacterial pneumonia remains an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Cytokines and chemokines are critical molecules expressed in response to invading pathogens and are necessary for normal lung bacterial host defenses. Here we show that interleukin (IL)-17, a novel cytokine produced largely by CD4+ T cells, is produced in a compartmentalized fashion in the lung after challenge with Klebsiella pneumoniae. Moreover, overexpression of IL-17 in the pulmonary compartment using a recombinant adenovirus encoding murine IL-17 (AdIL-17) resulted in the local induction of tumor necrosis factor-alpha, IL-1beta, macrophage inflammatory protein-2, and granulocyte colony-stimulating factor (G-CSF); augmented polymorphonuclear leukocyte recruitment; and enhanced bacterial clearance and survival after challenge with K. pneumoniae. However, simultaneous treatment with AdIL-17 provided no survival benefit after intranasal K. pneumoniae challenge. These data show that IL-17 may have a role in priming for enhanced chemokine and G-CSF production in the context of lung infection and that optimally timed gene therapy with IL-17 may augment host defense against bacterial pneumonia.

Animals↗

PubMed2001

CD4+ T cell-independent vaccination against Pneumocystis carinii in mice.

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. Moreover, deficient CD4+ T lymphocytes impair vaccination approaches to prevent opportunistic infection. Therefore, we investigated a CD4+ T cell-independent vaccine approach to a prototypic AIDS-defining infection, Pneumocystis carinii (PC) pneumonia. Here, we demonstrate that bone marrow-derived dendritic cells (DCs) expressing the murine CD40 ligand, when pulsed ex vivo by PC antigen, elicited significant titers of anti-PC IgG in CD4-deficient mice. Vaccinated animals demonstrated significant protection from PC infection, and this protection was the result of an effective humoral response, since adoptive transfer of CD4-depleted splenocytes or serum conferred this protection to CD4-deficient mice. Western blot analysis of PC antigen revealed that DC-vaccinated, CD4-deficient mice predominantly reacted to a 55-kDa PC antigen. These studies show promise for advances in CD4-independent vaccination against HIV-related pathogens.

Adoptive Transfer↗

PubMed2001

Prolonged ethanol treatment enhances lipopolysaccharide/phorbol myristate acetate-induced tumor necrosis factor-alpha production in human monocytic cells.

BACKGROUND: Ethanol (EtOH) is known to alter host immune responses and cytokine production. Acute EtOH exposure can suppress tumor necrosis factor (TNF)-alpha production, which attenuates pulmonary defense against infection. Previous studies in our laboratory show that acute EtOH inhibited TNF-alpha production by a posttranscriptional process, namely suppression of TNF-alpha-converting, enzyme-mediated, ectodomain shedding. However, chronic EtOH has been shown to augment TNF-alpha production, and this has been associated with EtOH-induced liver injury. To further characterize this paradoxical effect of EtOH on TNF-alpha production, we developed an in vitro model by using Mono Mac 6 cells, a human monocytic cell line. METHODS: Mono Mac 6 cells were treated with EtOH (0-75 mM) for 1 to 7 days. TNF-alpha production was induced by lipopolysaccharide and phorbol myristate acetate and quantitated by enzyme-linked immunosorbent assay. Generation of reactive oxygen species (ROS) was assayed by using a specific fluorogenic reagent. RESULTS: Acute EtOH initially inhibited lipopolysaccharide/phorbol myristate acetate-induced TNF-alpha production in Mono Mac 6 cells. However, during chronic EtOH exposure, this inhibition was reversed gradually over time. By day 6 after EtOH treatment, Mono Mac 6 cells demonstrated significant up-regulation of TNF-alpha production. Moreover, chronic EtOH induced the generation of ROS in these Mono Mac 6 cells. Scavenging ROS by Mn(III)tetrakis(1-methyl-4pyridyl)porphyrin pentachloride and N-acetyl-L-cysteine attenuated chronic EtOH-enhanced TNF-alpha production. CONCLUSION: These results suggest that ROS induction is involved in EtOH-enhanced TNF-alpha production by monocytes. This study also provides insight into the mechanisms of alteration of TNF-alpha production in different EtOH exposure settings.

Carcinogens↗

PubMed2000

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.

ADAM Proteins↗