Topical axitinib suppresses angiogenesis pathways induced by pulsed dye laser.

BACKGROUND: The recurrence of port-wine stain (PWS) blood vessels by pulsed dye laser (PDL)-induced angiogenesis is a critical barrier that must be overcome to achieve a better therapeutic outcome. OBJECTIVES: To determine whether PDL-induced angiogenesis can be suppressed by topical axitinib. METHODS: The mRNA expression profiles of 86 angiogenic genes and phosphorylation levels of extracellular signal regulated kinases (ERKs), phosphorylated protein kinase B (AKT) and ribosomal protein S6 kinase (p70S6K) in rodent skin were examined with or without topical axitinib administration after PDL exposure. RESULTS: The PDL-induced increased transcriptional levels of angiogenic genes peaked at days 3-7 post-PDL exposure. Topical application of 0·5% axitinib effectively suppressed the PDL-induced increase in mRNA levels of the examined angiogenic genes and activation of AKT, P70S6K and ERK from days 1 to 7 post-PDL exposure. After topical administration, axitinib penetrated into rodent skin to an approximate depth of 929·5 µm. CONCLUSIONS: Topical application of 0·5% axitinib can systematically suppress the PDL-induced early stages of angiogenesis via inhibition of the AKT/mammalian target of rapamycin/p70S6K and Src homology 2 domain containing transforming protein-1/mitogen-activated protein kinase kinase/ERK pathway cascades.

Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.

Idiopathic Pulmonary Fibrosis (IPF) is characterized by injury and loss of lung epithelial cells, accumulation of fibroblasts/myofibroblasts and abnormal remodeling of the lung parenchyma. The prognosis for IPF patients is poor and current therapies are largely ineffective in preventing respiratory failure. Current therapeutic approaches target epithelial cell replacement, manipulation of fibroblasts/myofibroblasts, modulation of procoagulant/fibrinolytic activities, cytokine and growth factor production, angiogenesis, and reduction of oxidative stress. Myofibroblasts are the primary effector cells in fibrosis. These cells may be derived by the activation and proliferation of resident lung fibroblasts, from epithelial-mesenchymal transition (EMT), or through recruitment of circulating fibrocytes. Transforming growth factor beta (TGFbeta) is a profibrotic factor that increases fibroblast proliferation, stimulates the synthesis and deposition of connective tissue, and inhibits connective tissue breakdown. TGFbeta acts through the promoter of the type 1 collagen gene causing increased collagen synthesis. In addition, TGFbeta induces EMT in alveolar epithelial cells (AECs) in vitro and in vivo. AECs exhibit substantial plasticity and may serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. Therapeutic interventions interfering with the pathways that lead to myofibroblast expansion and AEC apoptosis should be of considerable benefit in the treatment of IPF. This review will focus on the critical role of TGFbeta on AECs EMT and myofibroblasts in the development of fibrosis.

Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta.

Human endothelial cells produced a neutrophil chemotactic factor (NCF) upon stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS). The expression of endothelial cell-derived NCF messenger RNA and biological activity was both time- and concentration-dependent. Maximal NCF mRNA expression occurred at 10 and at 2 nanograms per milliliter for TNF and IL-1 beta, respectively; mRNA expression was first observed 1 hour after stimulation and was maintained for at least 24 hours. In situ hybridization analysis showed that NCF mRNA peaked in treated cells by 24 hours, whereas unstimulated cells were negative. These studies demonstrated that endothelial cells may participate in neutrophil-mediated inflammation by synthesizing a chemotactic factor in response to specific monokines and LPS.

Role of Smad3 in the regulation of rat telomerase reverse transcriptase by TGFbeta.

Telomerase is induced in certain pathological conditions such as cancer and tissue injury and repair. This induction in fibroblasts from injured lung is repressed by transforming growth factor beta (TGFbeta) via yet unknown mechanisms. In this study, the role of Smad3 in the inhibition of telomerase reverse transcriptase (TERT) gene transcription by TGFbeta was investigated. The rat TERT (rTERT) gene promoter was cloned by PCR amplification and fused with a luciferase reporter gene. This construct was used to analyse regulation of promoter activity in fibroblasts isolated from bleomycin-injured lung with induced telomerase activity. The results showed that TGFbeta inhibited rTERT transcription while stimulating Smad3 expression. Interestingly, TGFbeta also inhibited the expression of c-myc. Cotransfection with a Smad3 expressing plasmid further repressed rTERT transcription and c-myc expression, while cotransfection with the corresponding antisense Smad3 construct had the opposite effect. Mutation of an E-box in the rTERT promoter suppressed its activity, which could be further reduced by TGFbeta treatment. In contrast, mutation at a Smad binding element enhanced promoter activity whose inhibition was impaired by TGFbeta treatment. Thus TGFbeta inhibition of rTERT gene expression was directly mediated by Smad3 via the Smad binding element, while c-myc appears to primarily regulate its constitutive or induced expression.

Rat lung fibroblast collagen metabolism in bleomycin-induced pulmonary fibrosis.

Endotracheal bleomycin administration in rats and other animal species causes rapid development of pulmonary fibrosis, characterized by increased lung collagen synthesis and deposition. To clarify the mechanism, lung fibroblasts from bleomycin-treated rats (BRF) were isolated and maintained in tissue culture. They were then compared with those from normal untreated control animals, with respect to several key parameters of collagen metabolism. BRF synthesized collagen at a rate 35-82% above normal rat lung fibroblasts (NRF). This difference did not appear to be due to the selection of a clone by the subculture process. Furthermore, analysis of newly synthesized collagen type composition, revealed a significantly lower ratio of type III to type I collagen. Noncollagenous protein synthesis, however, was not significantly different from normal. Collagenase production and growth rate were also unaffected. BRF, however, was morphologically indistinguishable from NRF, even at the ultrastructural level. Upon further bleomycin (1 microgram/ml) exposure in vitro, BRF could be further stimulated to synthesize collagen at 82% above the rate for untreated BRF. This is comparable to the 90% increase in NRF treated in vitro (compared with untreated NRF). These results would favor the conclusion that bleomycin induces pulmonary fibrosis, by causing directly and/or indirectly lung fibroblasts (or a certain line of lung fibroblasts) to synthesize collagen at a higher rate without any associated increase in growth rate. The data, however, do not rule out the possibility that the fibroblast isolation procedure has selected for a certain population of fibroblasts that may not be typical of the in vivo situation.

Analysis of renal fibrosis in a rabbit model of crescentic nephritis.

The pathogenesis of renal fibrosis in crescentic nephritis is incompletely understood. To improve our understanding of this process, crescentic nephritis was induced in New Zealand White rabbits by administration of guinea pig antiglomerular basement membrane IgG after sensitization with guinea pig IgG, and their kidneys were analyzed for the development of fibrosis. Collagen synthesis in renal cortical tissue was significantly elevated by day 3, peaked at days 7-15, and returned towards baseline by day 21. Collagen content of both glomeruli and cortex were increased starting on days 14-16, and remained constant in cortex thereafter. Light microscopic analysis was much less sensitive, revealing fibrosis only after day 21. Immunofluorescence revealed that type IV collagen was distributed primarily in the glomerulus, while types I and III were increased in the glomerulus and interstitium. Thus, in this model of crescentic nephritis, fibrosis, as assessed biochemically, developed early at time points when morphologic analysis failed to detect such a development. Hence early therapeutic intervention, before morphologic evidence of fibrosis is evident, may be more successful in arresting the progression of this disease before it reaches irreversible terminal stages.

Stimulation of rat endothelial cell transforming growth factor-beta production by bleomycin.

This study examines the hypothesis that mediators from lung endothelial cells could promote lung collagen synthesis in pulmonary fibrosis. Since bleomycin induces pulmonary fibrosis in humans and animals, the effects of this drug on endothelial cells were examined. Endothelial cell conditioned media were prepared in the presence of various doses of bleomycin, and tested for their ability to stimulate lung fibroblast collagen synthesis. The results show a dose-dependent stimulation of endothelial cell secretion of collagen synthesis stimulatory activity by bleomycin, which peaked at a dose greater than or equal to 100 ng/ml. Stimulation was selective for collagenous protein synthesis. Gel filtration analysis showed most of the activity to reside in fractions with an estimated molecular mass range of 10-27 kD. The activity was inhibited by anti-transforming growth factor-beta (TGF-beta)antibody, but not by nonimmune control IgG. The presence of TGF-beta was confirmed using the mink lung epithelial cell assay. Northern blotting revealed significant increases in TGF-beta mRNA in bleomycin-stimulated endothelial cells. Thus in vitro stimulation of endothelial cells by bleomycin upregulates TGF-beta production, presumably by increased transcription. In view of the chemotactic and matrix synthesis stimulatory properties of this cytokine, such an increase in TGF-beta production may play an important role in bleomycin-induced pulmonary fibrosis.

Identification of an inducible regulator of c-myb expression during T-cell activation.

Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation.

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.

Decreased levels of 1,2-sn-diacylglycerol in human colon tumors.

We have found that in 15 of 15 primary human colon tumors there was a significant decrease (by about 40%) in the levels of diacylglycerol when compared to paired adjacent normal mucosa samples. Assays on the same samples indicated that this decrease was seen both in tumors that did and did not display mutations in codon 12 of c-K-ras. These results, taken together with previous studies on protein kinase C, suggest that the protein kinase C signal transduction pathway is suppressed in human colon cancer.

Human platelets mediate iron release from transferrin by adenine nucleotide-dependent and -independent mechanisms.

We assessed the ability of platelet sonicates and mediators secreted by unstimulated and thrombin-stimulated platelets to facilitate the release of iron from transferrin. Platelet sonicates and platelet conditioned media potentiated the release of iron from transferrin. The rate of release of iron was dependent on the pH of the reaction and amount of platelet sample added. Conditioned media from thrombin-stimulated platelets was more effective in mediating the release of iron from transferrin than was conditioned media from unstimulated cells. The rate of iron released from transferrin following addition of ATP and ADP in amounts equivalent to that present in platelet conditioned media was significantly less than the rate of iron released following the addition of conditioned media from platelets. Depletion of ATP and ADP in platelet conditioned media by incubation with apyrase only partially inhibited their ability to enhance the rate of iron release from transferrin. These observations indicate that platelets enhance the release of iron from transferrin by adenine nucleotide-dependent and -independent mechanisms. These observations are consistent with the hypothesis that platelets promote oxidant-induced tissue injury at sights of inflammation secondary to their ability to enhance the local release of iron from transferrin.

Prostaglandin modulation of N-formylmethionylleucylphenylalanine-induced transmembrane potential changes in rat neutrophils.

Prostaglandins of the E-series (PGEs) and PGI2 will inhibit formylmethionylleucylphenylalanine-(f-Met-Leu-Phe) induced lysosomal enzyme release and superoxide-anion (O2-) production by neutrophils. The inhibitory effects of PGEs and PGI2 on neutrophil functional responses have been correlated with their ability to increase intracellular cAMP. In this study we have examined the effects of PGEs and PGI2 on f-Met-Leu-Phe- and phorbol-myristate-acetate-induced rat neutrophil membrane potential changes using an optical probe of membrane potential 3,3-dipropylthiodicarbocyanine iodide. 15-(S)-15-methyl-PGE1 (15-methyl-PGE1), a stable analogue of PGE1 and PGI2 inhibited f-Met-Leu-Phe-induced transmembrane potential changes in a dose-dependent manner. This inhibition was correlated with the ability of these agents to increase intracellular cAMP levels and inhibit O2- production and degranulation. In contrast, 15-methyl-PGE1 and PGI2, did not inhibit phorbol-myristate-acetate-induced transmembrane potential changes and O2- production. These results suggest independent mechanisms of activation of neutrophils by phorbol myristate acetate and f-Met-Leu-Phe, and they also suggest that the inhibitory effects of prostaglandins and cAMP on f-Met-Leu-Phe-stimulated cells is at a step or steps prior to activation of those processes involved in effecting changes in transmembrane potential, which are common to both stimuli.

Superoxide dismutase does not cause scar thinning after myocardial infarction.

Previous studies demonstrated that treatment with superoxide dismutase, a scavenger of superoxide anions, limits the extent of myocardial injury in a canine preparation of regional myocardial ischemia and reperfusion. Little is known, however, about the effects of superoxide dismutase on the healing of a myocardial infarct. Therefore, this study was performed to determine whether treatment with superoxide dismutase during myocardial ischemia impairs formation of scar tissue after infarction. Dogs received 2 hour infusions of superoxide dismutase or albumin (controls) by way of the left atrium beginning 15 minutes before and ending 15 minutes after a 90 minute occlusion of the left circumflex coronary artery. Six weeks later the animals were killed. Two-dimensional echocardiography was performed before surgery and before induced death. Wall thickening in the central ischemic zone was decreased at 6 weeks compared with baseline studies (p less than 0.05), but the decrease was similar for both groups. The hydroxyproline concentrations (microgram/mg dry weight) of the scar tissue in the superoxide dismutase and control groups, respectively, were 35.3 +/- 3.8 and 28.7 +/- 5.0 (p less than 0.05). The ratios of the scar thickness to normal wall thickness were superoxide dismutase 0.91 +/- 0.03 and control 0.89 +/- 0.03 (p greater than 0.05). Thus, superoxide dismutase had no adverse effect on wall thickening or scar formation assessed 6 weeks after myocardial infarction, and may be useful to limit oxygen radical-mediated damage during reperfusion of the ischemic myocardium.

Molecular mechanisms of and possible treatment strategies for idiopathic pulmonary fibrosis.

Pulmonary fibrosis is characterized by lung inflammation and abnormal tissue repair, resulting in the replacement of normal functional tissue with an abnormal accumulation of fibroblasts and deposition of collagen in the lung. This process involves cellular interactions via a complex cytokine-signaling mechanism and heightened collagen gene expression, ultimately resulting in its abnormal collagen deposition in the lung. Our current understanding of the pathogenesis of pulmonary fibrosis suggests that in addition to inflammatory cells, the fibroblast and signaling events that mediate fibroblast proliferation and myofibroblasts, play important roles in the diverse processes that constitute fibrosis. Increasing knowledge of cytokine biology, cytokine-signaling and cell matrix interactions have shed some light on the genesis of pulmonary fibrosis; however, the importance of inflammation in pulmonary fibrosis remains controversial. This remains true because the inflammatory component is variable at the time of diagnosis, and the most potent anti-inflammatory drugs that have been widely used in the treatment of pulmonary fibrosis do not seem to interfere with the fibrotic disease progression. Pulmonary fibrosis is a highly lethal disorder, which continues to pose major clinical challenges because an effective therapeutic regimen is yet to be determined. This review summarizes recent progress in understanding the molecular mechanisms of pulmonary fibrosis, and includes a more detailed discussion of the potential points of therapeutic attack in pulmonary fibrosis. In addition, a detailed discussion is presented regarding each of the potential therapies which have emerged from the animal models of pulmonary fibrosis, and which have been developed through advances in cellular and molecular biology.

Bleomycin binding sites on alveolar macrophages.

Previous work has demonstrated that bleomycin can directly stimulate alveolar macrophage secretion of fibroblast growth factors and monocyte chemotactic factors. In this study, rat alveolar macrophages obtained by bronchoalveolar lavage were examined for the presence of bleomycin binding sites, which might mediate this response. The results indicated that alveolar macrophages have specific, saturable, and reversible binding sites. Both high- and low-affinity binding sites were found; each macrophage possessed 6.7 x 10(4) high-affinity sites, with a Kd of 528 nM, and 2.2 x 10(6) low-affinity sites, with a Kd of 65 microM. The Kd of the high-affinity sites corresponds closely to the ED50 obtained from dose-response curves of the bleomycin-stimulated secretion of both fibroblast growth and monocyte chemotactic factors, suggesting that bleomycin stimulation of alveolar macrophage function responses may be mediated by bleomycin interaction with these sites.

TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury.

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.

The role of IL-5 in bleomycin-induced pulmonary fibrosis.

Eosinophils are known to express cytokines capable of promoting fibrosis. Interleukin-5 (IL-5) is important in regulating eosinophilopoiesis, eosinophil recruitment and activation. Lung IL-5 expression is elevated in pulmonary fibrosis, wherein the eosinophil is a primary source of fibrogenic cytokines. To determine the role of IL-5 in pulmonary fibrosis, the effects of anti-IL-5 antibody were investigated in a model of bleomycin-induced pulmonary fibrosis. Fibrosis was induced in mice by endotracheal bleomycin treatment. Animals were also treated with either anti-IL-5 antibody or control IgG. Lungs were then analyzed for fibrosis, eosinophil influx, chemotactic activity, and cytokine expression. The results show that a primary chemotactic activity at the height of eosinophil recruitment is IL-5. Furthermore, anti-IL-5 antibody caused significant reduction in lung eosinophilia, cytokine expression, and fibrosis. These findings taken together suggest an important role for IL-5 in pulmonary fibrosis via its ability to regulate eosinophilic inflammation, and thus eosinophil-dependent fibrogenic cytokine production.

Regulation of macrophage-derived fibroblast growth factor release by arachidonate metabolites.

The macrophage is a source of many mediators with direct and indirect fibrogenic potential. In this study, release of macrophage-derived fibroblast growth factor (MDGF) activity by murine peritoneal macrophages is examined with regard to its regulation by arachidonate metabolites. Upon stimulation with 10 micrograms/ml lipopolysaccharide (LPS), resident peritoneal macrophages from CBA/J mice released MDGF activity into media rapidly, reaching maximal levels in approximately 1 h. Lysates of these stimulated cells also revealed significantly increased cell-associated MDGF activity, composing 45% of the total assayable activity. This activity, as assayed by radioactive thymidine incorporation by primary cultures of rat lung fibroblasts, was separable from interleukin-1 (IL-1) activity by reverse phase high performance liquid chromatography (HPLC). Furthermore, purified murine IL-1 had no MDGF activity in this assay system. This stimulated MDGF release was enhanced by the cyclooxygenase inhibitors indomethacin, ibuprofen, and aspirin at micromolar concentrations, but inhibited in a dose-dependent manner by prostaglandin E2 (PGE2). On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor was inhibitory at 0.1 and 0.4 microM but not at 2.5 microM. Zymosan-stimulated macrophages also markedly increased MDGF release, albeit with a different time course which was characterized by a delay of approximately 7 h before peak levels were attained. Such stimulation, which is known to cause increased lipoxygenase activity, was also inhibited by 0.5 microM NDGA. In contrast, the lipoxygenase pathway products leukotrienes B4 (LTB4) and C4 (LTC4) stimulated MDGF release in a dose-dependent (10(-10)-10(-8) M) manner, with LTC4 being more potent on a per unit dose basis. Stimulation by LTC4 was inhibited by the putative leukotriene receptor antagonist, FPL55712, while LTD4 and LTE4 did not stimulate MDGF release, thus suggesting the mediation of this effect by specific LTC4 receptors. These data suggest also that products of the cyclooxygenase and lipoxygenase pathways are potentially important both as exogenous (ie, derived from cells other than the macrophage itself) and auto- or self-regulators of macrophage MDGF release. This, in turn, implies that cyclooxygenase products are antifibrogenic and important in maintaining or returning to the quiescent or normal state, whereas the lipoxygenase products are profibrogenic and important in induction of fibrosis or wound-healing and tissue repair. Any alteration in the balance between these two pathways may result in either a desirable or a harmful outcome.

3-deaza-adenosine inhibition of stimulus-response coupling in human polymorphonuclear leukocytes.

In an effort to define better the functional role of S-adenosyl-methionine-mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3-deaza-adenosine (3-DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl-leucyl-phenylalanine (FMLP)-induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3'-dipropylthiocarbocyanine (di-S-C3-(5)) as an optical probe of membrane potential we observed that 3-DZA at concentrations that inhibit FMLP-induced O2- production does not significantly alter FMLP-induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3-DZA on FMLP-induced PMN pinocytosis and to a lesser degree on FMLP-induced degranulation. However, pretreatment of PMNs with 3-DZA did not alter FMLP-induced changes in Quin-2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor-induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor-induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor-induced methylation reactions. Furthermore, 3-DZA did not alter phorbol myristate acetate induced O2- production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2- production.

A role for C-C chemokines in fibrotic lung disease.

Pulmonary fibrosis is the end point of a chronic inflammatory process characterized by leukocyte recruitment and activation, fibroblast proliferation, and increased extracellular matrix production. Previous studies of models of pulmonary fibrosis have investigated the role of cytokines in the evolution of the fibrotic response. The involvement of tumor necrosis factor and interleukin-1 in bleomycin-induced lung injury, a model of idiopathic pulmonary fibrosis, has been well established, suggesting that cytokines mediate the initiation and maintenance of chronic inflammatory lesions. However, the aforementioned cytokines alone cannot account for the recruitment and activation of specific leukocyte populations found in the bleomycin model. Recently, a family of novel proinflammatory cytokines (chemokines) was cloned and characterized, yielding many putative mediators of leukocyte functions. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemoattractant protein-1 (MCP-1) belong to the C-C chemotactic cytokine family, a group of low-molecular-weight peptides. These molecules modulate chemotaxis, proliferation, and cytokine expression in leukocyte subsets. Our group has investigated the roles of MCP-1 and MIP-1 alpha in the bleomycin model. Both MCP-1 and MIP-1 alpha are expressed in a time-dependent manner after bleomycin challenge, and passive immunization of these animals with either anti-MIP-1 alpha or anti-MCP-1 antibodies attenuated leukocyte accumulation. In addition, we have identified specific cell types expressing MCP-1 or MIP-1 alpha by in situ hybridization and immunohistochemical localization, respectively. Furthermore, our results indicate that MIP-1 alpha expression is mediated by alveolar macrophage-derived tumor necrosis factor, identifying an important cytokine pathway in the initiation of pulmonary fibrosis. Finally, anti-MIP-1 alpha therapy attenuated fibrosis, providing direct evidence for its involvement in fibrotic pathology. Our work has clearly established that the C-C chemokines MCP-1 and MIP-1 alpha are expressed and contribute to the initiation and maintenance of the bleomycin-induced pulmonary lesion.