Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity

We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-beta Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-beta in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.     

Mycolyl glycolipids stimulate macrophages to release a chemotactic factor

We have previously reported that mycolyl glycolipids from Nocardia rubra such as glucose or trehalose mycolates induced granuloma formation in mice. The structure of the carbohydrate moiety of the mycolyl glycolipids influenced the granuloma forming activity profoundly. Here, we have examined the macrophage-chemotactic activity in the culture supernatants stimulated with various glycolipids differing in carbohydrate moiety (trehalose 6,6'-dimycolate, or TDM; glucose monomycolate, or GM; mannose monomycolate, or MM; and fructose monomycolate, or FM). A distinctive chemotactic activity was detected with TDM or GM, but, little or none with MM or FM.     

Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity

Weinvestigated a role of neuroregulation in the release of eosinophilchemotactic activity (ECA) from bovine bronchial epithelial cells(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P(SP), and the supernatant fluids were tested for ECA by a blind-wellchemotactic chamber technique. BBEC released ECA in response to ACh andSP in a dose- and time-dependent manner. Checkerboard analysis showedthat ECA in regard to ACh and SP was chemotactic rather thanchemokinetic. Partial characterization revealed that ECA involved bothlipids and peptides. The release of ECA in response to ACh and SP wasinhibited by nonspecific and 5-specific lipoxygenase inhibitors and bycycloheximide (P < 0.01). Molecular-sieve columnchromatography revealed that these mediators induced three molecularmass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowestpeak, which represented the predominant activity, was blocked byleukotriene B₄-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The releaseof leukotriene B₄ in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).Platelet-activating factor was not detected. These results raise thepossibility of a role of neuroregulation for the elaboration of ECA inthe airway.

     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. A potential mechanism for neutrophil recruitment, cytotoxicity, and inhibition of proliferation in bronchial inflammation.

To test the effect of endotoxin on bronchial epithelial cells (BEC), BEC were isolated from bovine lungs and cultured in the presence of bacterial endotoxin. The BEC culture supernatant fluids were harvested, and neutrophil chemotactic activity (NCA) was determined with a blindwell chamber technique; cytotoxicity determined by lactate dehydrogenase release and BEC proliferation determined by Coulter counting. Endotoxin caused a dose- and time-dependent release of NCA from BEC cultures compared with media alone (82.3 +/- 8.1 vs 12.0 +/- 3.1 cells/high power field, p less than 0.001). To further characterize this activity, reverse phase HPLC analysis of release eicosanoid metabolites after [3H]arachidonic acid incorporation was performed. Endotoxin stimulated the release of the neutrophil chemoattractants, leukotriene B4 and 12-hydroxyeicosatetraenoic acids. Endotoxin also resulted in a dose and time dependent release of lactate dehydrogenase (42.9 +/- 4.2 vs 20.2 +/- 2.2 U/liter, p less than 0.001) although higher doses were required to cause cytotoxicity than to stimulate chemotaxis. Finally, endotoxin resulted in a dose dependent inhibition of BEC proliferation (176 x 10(3) +/- 16 x 10(3) vs 1,080 x 10(3) +/- 38 x 10(3) cells/ml measured at day 14, p less than 0.001). These data suggest that bacterial release of endotoxin may contribute to the pathophysiologic changes observed in bronchial inflammation by stimulating BEC to release NCA, denuding airway epithelium by causing cytotoxicity of BEC, and inhibiting epithelial repair by inhibiting BEC proliferation.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.     

Inhibition of the eosinophil chemotactic factor (ECF) release from human PMNs and rat mast cells by arachidonic acid analogs

An eosinophil chemotactic factor (ECF) can be released from human polymorphonuclear neutrophils (PMN) and rat mast cells by the calcium ionophore A23187, during phagocytosis, by arachidonic acid and melittin. It has been suggested that these stimuli lead to phospholipid turnover with the generation of arachidonic acid, which is subsequently transformed by a converting enzyme to ECF. Addition of polienoic (5,8,11-eicosatrienoic and 4,7,10,13-eicosatetraenoic acids) or poliynoic acids (5,8,11-eicosatriynoic and 4,7,10,13-eicosatetraynoic acids) induced a dose- and time-dependent inhibition of ECF release from the cells. Poliynoic acids are more potent inhibitors than polienoic acids. Among the former 4,7,10,13-eicosatetraynoic acid is the most effective substance.     

Enhanced release of oxygen metabolites by monocyte-derived macrophages exposed to proteolytic enzymes: activity of neutrophil elastase and cathepsin G

Macrophages at sites of inflammation are exposed to proteolytic enzymes derived from neutrophils, platelets, clotting factors, complement, and damaged tissues. To investigate the possible effect of proteases on the plasma membrane-mediated oxidative metabolic response of macrophages in inflammatory sites, cultured human monocyte-derived macrophages were treated in vitro with proteolytic enzymes and were then assayed for their ability to release superoxide anion (O2-) and hydrogen peroxide (H2O2) in response to stimuli. Macrophages pretreated for 1 to 20 min with trypsin, chymotrypsin, pronase, or papain, 0.1 to 200 micrograms/ml, released up to 3.5-times more O2- and H2O2 than did control (untreated) cells. This enhanced production of oxygen metabolites was observed by using either phorbol myristate acetate or opsonized zymosan as the stimulus. Macrophages were also "primed" for enhanced O2- release (2.3-fold) by pretreatment with a subfraction of granules extracted from human neutrophils. This subfraction contained primarily elastase and cathepsin G. Similar enhancement was observed with 60 ng/ml or purified human neutrophil cathepsin G (2.2-fold) and with 20 micrograms/ml of purified neutrophil elastase (3.3-fold). Priming by these neutrophil proteases could be blocked by specific inhibitors of their proteolytic activity. These results suggest that macrophages involved in an inflammatory response might be rapidly primed by proteases released from degranulating neutrophils. Primed macrophages could mount a more effective oxidative metabolic response to microorganisms or tumor cells, but might also cause greater tissue damage.     

Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway.

This study investigates whether the guanine nucleotide exchange activity of Vav is linked to cytokine production in mast cells. Overexpression of Vav in the RBL-2H3 mast cell line resulted in the constitutive tyrosine phosphorylation and activation of Vav. We analyzed the functional effect of Vav overexpression on cytokine production. IL-2 and IL-6 mRNA levels were dramatically increased in Vav-overexpressing cells and correlated with increased NF-AT activity. Little or no effect was observed on the mRNA levels of IL-3, IL-4, GM-CSF, TNF-alpha, and TGF-beta. FcepsilonRI engagement did not further enhance IL-2 and IL-6 mRNA levels and only slightly enhanced NF-AT activity, but dramatically increased the mRNA levels of other tested cytokines. To understand the signal transduction required, we focused primarily on IL-6 induction by measuring mitogen-activated protein kinase activity and analyzing the effects of mutant or dominant negative forms of Vav, Rac1, and c-Jun N-terminal kinase-1 (JNK1). Vav overexpression resulted in the constitutive activation of JNK1 with little or no effect on p38 mitogen-activated protein kinase and ERK2. This was dependent on Vav-mediated activation of Rac1 as a Dbl domain-mutated Vav, inactive Rac N17, and inactive JNK1 down-regulated the Vav-induced JNK1 or IL-6 responses. Vav expression, but not expression of domain-mutated Vav, increased IL-6 secretion from nonimmortalized bone marrow-derived mast cells upon FcepsilonRI engagement. We conclude that Vav phosphorylation contributes to IL-6 induction in mast cells.     

Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T cells

The cytokines secreted by pathogen-activated human dendritic cells (DC) are strongly regulated in vitro by histamine, a major component of mast cell granules, ultimately modulating the capacity of the DC to polarize naive T cells. Because DC and mast cells are located in close proximity in peripheral compartments, we hypothesized that mast cell products would influence the maturation of DC and hence the Th balance of an immune response in vivo. In this study, we show that specific mast cell degranulation stimuli, given s.c. in mice with Ag and adjuvant, produce effector T cells that proliferate to Ag but secrete dramatically reduced levels of IFN-gamma and increased amounts of IL-4 compared with control T cells primed in the absence of a mast cell stimulus. Immunization with Ag and adjuvant in the presence of a degranulation stimulus also resulted in the accumulation of DC in the draining lymph nodes that had reduced capacity to induce Ag-specific Th1 cells, in comparison with DC from mice lacking a degranulation stimulus. Therefore, by acting upon DC at sites of inflammation, mast cells play a critical role in determining the polarity of Ag-specific T cell responses in vivo.     

Lipid bodies: cytoplasmic organelles important to arachidonate metabolism in macrophages and mast cells

Much has been learned about the biochemical nature and pharmacologic activity of the products of arachidonic acid (AA) oxidation, but relatively little is known about the structures in nucleated cells into which AA is incorporated and from which it is initially mobilized. To address this question, we administered 3H-AA or other 3H-fatty acids in vitro to human lung mast cells and alveolar macrophages as well as to mouse and guinea pig peritoneal macrophages. The subcellular distribution of 3H label was assessed by electron microscopic autoradiography, and the nature of cell-associated 3H-lipids was determined by thin layer chromatography. Autoradiographic analysis of human lung mast cells localized virtually all of the 3H-AA to cytoplasmic lipid bodies. Lipid bodies are roughly spherical, variable osmiophilic, nonmembrane-bound structures that appear in the cytoplasm of a wide variety of cells, but we have found that these lipid bodies occur with increased frequency in granulocytes, macrophages, and mast cells at sites of inflammatory, immunologic, or neoplastic processes. Macrophages also incorporated 3H-AA predominantly into cytoplasmic lipid bodies. In contrast to mast cells, however, macrophages incorporated 3H-AA into the plasma membrane as well. Stimulation of macrophage phagocytosis resulted in striking alterations of the relationships of lipid bodies to intracellular membranes, so that many lipid bodies appeared adjacent to phagolysosomes. In addition, some phagolysosomes contained 3H label, which along with other morphologic evidence suggested that lipid bodies may discharge their contents into these structures. Mast cell and macrophage cytoplasmic lipid bodies appear to represent a major site of intracellular storage and metabolism of products of AA and perhaps other fatty acids taken up from the external milieu. These heretofore neglected organelles may thus influence cellular function in a wide variety of adaptive or pathologic processes.     

Antirheumatic drug effects on neutrophil response to chemotactic factors: a comparison of analytical techniques

A recently reported technique employing the leukotactic index which represents all migrating cells in vitro neutrophil chemotaxis systems, was compared to the leading front technique for assaying antirheumatic drug effects on this important neutrophil function. Normal human neutrophils were treated with therapeutic concentrations of aspirin, gold sodium thiomalate, D-penicillamine, and azathioprine. The responses of these cells and of control cells to neutrophil-immune complex-serum-derived chemotactic factors were assayed in Boyden chambers. Significant (P less than 0.05) inhibition was observed by the leading front technique only for D-penicillamine at high concentrations. Significant (P less than 0.01) inhibition was seen with D-penicillamine at therapeutic plasma levels with the leukotactic index technique. Gold sodium thiomalate and aspirin at high concentrations also produced significant (P less than 0.01 and P less than 0.05) inhibition of chemotaxis as assayed by the leukotactic index procedure. Azathioprine had no significant effects when studied with either technique. These results indicate that the leukotactic index may be a more sensitive technique for quantitating neutrophil migration in response to chemotactic factors and may therefore provide useful additional information for determining the effects of antirheumatic drugs on this important neutrophil function.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: Evidence for a prostacyclin-dependent mechanism

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (Mø) in vitro. AA (0.5–16 μM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 μM AA generating a peak of IL-6 release (3-5-fold). AA (0.5–16 μM) also induced an increasing release of the AA metabolite thromboxane B₂ (TXB₂). The AA-induced release of IL-6 occurred within 1–2 h of incubation, whereas TXB₂ concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 μM and 40.0 μM) effectively blocked the generation of TXB₂, but increased prostacyclin (PGI₂) generation and potentiated the release of IL-6. In addition, PGI₂, as well as the PGI₂ agonists iloprost and cicaprost, stimulated IL-6 release from Mø by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI₂ (but not TXA₂) is involved in AA-induced IL-6 release from peritoneal Mø.     

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.     

Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.