Release Of Chemotactic Activity Research Articles

Identification and Characterization of Circulating Variants of CXCL12 from Human Plasma: Effects on Chemotaxis and Mobilization of Hematopoietic Stem and Progenitor Cells

Mobilization of hematopoietic stem and progenitor cells (HPCs) is induced by treatment with granulocyte-colony stimulating factor, chemotherapy, or irradiation. We observed that these treatments are accompanied by a release of chemotactic activity into the blood. This plasma activity is derived from the bone marrow, liver, and spleen and acts on HPCs via the chemokine receptor CXCR4. A human blood peptide library was used to characterize CXCR4-activating compounds. We identified CXCL12[22-88] and N-terminally truncated variants CXCL12[24-88], CXCL12[25-88], CXCL12[27-88], and CXCL12[29-88]. Only CXCL12[22-88] could effectively bind to CXCR4 and induce intracellular calcium flux and chemotactic migration of HPCs. CXCL12[25-88] and CXCL12[27-88] revealed neither agonistic nor antagonistic activities in vitro, whereas CXCL12[29-88] inhibited CXCL12[22-88]-induced chemotactic migration. Since binding to glycosaminoglycans (GAG) modulates the function of CXCL12, binding to heparin was analyzed. Surface plasmon resonance kinetic analysis showed that N-terminal truncation of Arg22-Pro23 increased the dissociation constant KD by one log10 stage ([22-88]: KD: 5.4 ± 2.6 μM; [24-88]: KD: 54 ± 22.4 μM). Further truncation of the N-terminus decreased the KD ([25-88] KD: 30 ± 4.8 μM; [27-88] KD: 23 ± 1.6 μM; [29-88] KD: 19 ± 5.4 μM), indicating increasing competition for heparin binding. Systemic in vivo application of CXCL12[22-88] as well as CXCL12[27-88] or CXCL12[29-88] induced a significant mobilization of HPCs in mice. Our findings indicate that plasma-derived CXCL12 variants may contribute to the regulation of HPC mobilization by modulating the binding of CXCL12[22-88] to GAGs rather than blocking the CXCR4 receptor and, therefore, may have a contributing role in HPC mobilization.

METHOTREXATE STIMULATES LUNG EPITHELIAL CELLS TO RELEASE INFLAMMATORY CELL CHEMOTACTIC ACTIVITIES

Methotrexate-induced pneumonitis has been reported as an infrequent but potentially serious complication of therapy in a variety of malignant and benignconditions. Because inflammatorycell infiltration is concerned with the development of methotrexate-induced pneumoinitis, and because airway epithelial cells participate in the orchestration of lung inflammation, the authors determined whether methotrexate might stimulate airway epithelial cells (A549 cells) to release neutrophil, monocyte, and eosinophil chemotactic activities (NCA, MCA, and ECA). A549 cells released NCA, MCA, and ECA in a dose- and time-dependent manner in response to methotrexate. Partial characterization revealed the heterogeneity of NCA, MCA, and ECA. The release of chemotactic activity was blocked by lipoxygenase inhibitors and cycloheximide. NCA was inhibited by leukotriene (LT) B 4 receptor antagonist, and anti-interleukin (IL)-8 and granulocyte colony-stimulatingfactor (G-CSF) antibodies. MCA was attenuated by LTB 4 receptor antagonist, and anti-monocyte chemoattractant protein (MCP)-1 and granulocyte-macrophage CSF (GM-CSF) antibodies. ECA was attenuated by LTB 4 receptor antagonist, and anti-IL-8 and GM-CSF antibodies. The release of IL-8, G-CSF, MCP-1, GM-CSF, and LTB 4 from A549 cells significantly increased in response to methotrexate. The mRNA expression of IL-8 and MCP-1 was augmented by methotrexate stimulation. These data suggest that type II epithelial cells may modulate inflammatory cell recruitment into the lung by releasing NCA, MCA, and ECA in response to methotrexate.

Cyclophosphamide stimulates lung fibroblasts to release neutrophil and monocyte chemoattractants.

Cyclophosphamide is an alkylating antineoplastic agent used in several conditions. However, little is known about the mechanism of its pulmonary toxicity. In the present study, we determined that human lung fibroblasts release activity for neutrophils and monocytes in response to cyclophosphamide in a dose- and time-dependent manner. Checkerboard analysis revealed that both neutrophil and monocyte activities were chemotactic. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular-sieve column chromatography revealed that both neutrophil (NCA) and monocyte (MCA) chemotactic activities had multiple peaks. NCA was inhibited by a leukotriene B(4) receptor antagonist and anti-interleukin-8 and anti-granulocyte colony-stimulating factor antibodies. MCA was attenuated by a leukotriene B(4) receptor antagonist and anti-monocyte chemoattractant protein-1 and anti-granulocyte-macrophage colony-stimulating factor antibodies. The concentrations of interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor significantly increased in response to cyclophosphamide. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to cyclophosphamide.

Bradykinin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity.

Activation of the kallikrein-kinin system in lung injury has long been recognized. However, the effects of bradykinin (BK) on human lung fibroblasts (HLF) remain to be elucidated. We determined whether BK stimulates HLF to release chemotactic activity for neutrophils and monocytes (NCA and MCA, respectively). We evaluated HLF supernatant fluids for chemotactic activity through a blind-well chamber technique. HLF released NCA and MCA in a dose- and time-dependent manner in response to BK. 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 a leukotriene (LT) B(4) receptor antagonist and by antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF). MCA was attenuated by the LTB(4) receptor antagonist and by antibodies to monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-beta. Both the LTB(4) receptor antagonist and these antibodies inhibited chemotactic activity of the molecular weights corresponding to MCP-1, GM-CSF, and TGF-beta, separated by column chromatography. The concentrations of IL-8, G-CSF, MCP-1, GM-CSF, and TGF-beta in supernatant fluids increased significantly in a time-dependent manner in response to BK. The receptors responsible for the release of NCA, MCA, and individual chemokines included both BKB(1) and BKB(2) receptors. These data suggest that BK may stimulate lung fibroblasts to release inflammatory cytokines, which may modulate lung inflammation.

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.

Complement-induced release of monocyte chemotactic protein-1 from human smooth muscle cells. A possible initiating event in atherosclerotic lesion formation.

Increasing evidence suggests that complement activation might represent an important mechanism in early atherogenesis. Thus, complement components, in particular the membrane attack complex (MAC) C5b-9(m), have been isolated from human atherosclerotic lesions. Furthermore, complement activation is known to occur in atherosclerotic lesions induced in experimental animals, and the severity of cholesterol-induced plaques is markedly reduced in complement-deficient animals. During atherogenesis monocytes are recruited into the arterial wall, and a potent chemoattractant for monocytes, monocyte chemotactic protein-1 (MCP-1), is expressed by vascular smooth muscle cells (SMCs). We hypothesized that generation of MACs on SMCs during the activation of complement might lead to the release of MCP-1 and hence to monocyte recruitment. In this study, MACs were generated on human SMCs in vitro by sequential addition of the purified complement components C5b6, C7, C8, and C9. This supernatant of the culture was chemotactic for freshly isolated peripheral blood monocytes in a modified Boyden chamber. The chemotactic activity of the supernatant was abolished by anti-MCP-1 blocking antibodies but not by an isotype-matched antibody against an irrelevant antigen. The release of chemotactic activity was dependent on the dose of MAC formed on SMCs and was demonstrated within 10 minutes of exposure of the cells. The data support the hypothesis that complement-mediated release of MCP-1 from SMCs might be important in the recruitment of monocytes into the developing atherosclerotic lesion and could be an important initiating event in atherogenesis.

Cigarette Smoke Stimulates Release of Neutrophil Chemotactic Activity from Cultured Bovine Bronchial Epithelial Cells

1. Recruitment of neutrophils into the airway is a prominent feature of chronic bronchitis, a syndrome often associated with exposure to cigarette smoke. Since bronchial epithelial cells are the 'first' lung cells to come into contact with smoke, these cells may be responsible for neutrophil recruitment into the airway by release of neutrophil chemotactic activity in response to cigarette smoke. 2. To evaluate this hypothesis, we prepared bovine bronchial epithelial cells and measured their ability to release neutrophil chemotactic activity following exposure to cigarette smoke. Bronchial epithelial cells were prepared by overnight digestion with protease, filtered through 100-microns Nitex mesh and resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and antibiotics and cultured at 2 x 10(6) cells in 2 ml of medium in 35-mm culture dishes. After 4 days, dishes were rinsed and refed with medium without fetal calf serum and incubated with and without 1:10 diluted smoke extract for 6 h at 37 degrees C. The neutrophil chemotactic activity of the supernatant fluids was measured by the blindwell chamber technique. 3. Cigarette smoke itself added to medium did not stimulate chemotaxis. In contrast, cigarette smoke did stimulate the release of neutrophil chemotactic activity from bovine bronchial epithelial cells [15 +/- 3 (control) versus 74 +/- 5 (smoke), P < 0.01]. 4. This neutrophil chemotactic activity was dialysable, pepsin and acid stable, heat sensitive and lipid extractable. Sephadex G-75 column chromatography demonstrated two peaks of neutrophil chemotactic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil chemotaxis to extracts of grain plant components

Exposure to grain dust has been associated with a neutrophilic bronchitis. In vitro studies suggest several possible mechanisms for recruitment of neutrophils, including direct chemotaxis to aqueous grain dust extracts and release of chemotactic activity by bronchial epithelial cells in response to challenge with grain dust extracts.

Generation of lipid neutrophil chemoattractant by irradiated bovine aortic endothelial cells.

Radiation injury to blood vessels is associated with an acute inflammatory process. We investigated the capacity of cultured bovine aortic endothelial cells (BAEC) to produce chemotactic factors after radiation injury. BAEC in serum-free media were irradiated with a cobalt-60 Gammacell 220 and the cell supernatants were assayed for chemotactic activity for human neutrophils in a Boyden chamber. There was a rapid release of chemotactic activity into the BAEC supernatants which was dependent both on the dose of radiation (5 to 40 Gy) and the time between irradiation and sample collection. In contrast, isolation of BAEC lysates by freeze-thawing was not associated with the presence of similar chemotactic activity. The chemotactic activity released from the irradiated BAEC was not destroyed by boiling nor by treatment with trypsin. The release of the chemotactic activity was, however, inhibited by the addition of a lipoxygenase inhibitor but not by the addition of a cyclooxygenase inhibitor before the irradiation. The chemotactic activity was recovered from the cell supernatants in the lipid phase after extraction with chloroform/methanol. Furthermore, the chloroform/methanol extracts co-eluted with authentic leukotriene B4 when the BAEC were prelabeled with [14C] arachidonic acid. However, we were unable to detect endogenous leukotriene B4 with RIA. Instead, the only detectable endogenous lipid present in the supernatants was 13-hydroxyoctadecadienoic acid which is derived from linoleic acid via the lipoxygenase pathway. 13-Hydroxyoctadecadienoic acid, however, had no chemotactic activity. These findings suggest that endothelial cells rapidly release a chemotactic agent after irradiation, the release of which is associated with a lipoxygenase pathway. The release of this chemotactic activity may account in part for the acute inflammatory response that is observed after ionizing irradiation.

Benign Asbestos Pleurisy in the Rabbit

Benign asbestos pleurisy is a manifestation of asbestos-induced disease that is not uncommon but often ignored. We developed an animal model to study the pathogenesis of this syndrome. Crocidolite asbestos injected into the rabbit pleural space resulted in the appearance of chemotactic activity in an exudative effusion, characterized by a polymorphonuclear leukocyte (PMN) response that peaked 4 h after the injection. The chemotactic activity and the PMN responses occurred equally in normal animals and in animals that had been depleted of complement with cobra venom. Neutropenia, induced with vinblastine, did not alter the appearance of chemotactic activity but abrogated the PMN response. Treatment with colchicine failed to block the release of chemotactic activity into the pleural fluid, but decreased the PMN response without affecting the ability of peripheral blood PMN to respond to pleural fluid chemotactic activity. When pleural fluid containing chemotactic activity was injected into the pleural space of another rabbit, a PMN response was observed. These studies suggest that the acute exudative response seen in the rabbit model of benign asbestos pleurisy results from the release of chemotactic activity from sources other than complement. The response probably depends on interaction between the asbestos and the pleural tissue. Asbestos-PMN interaction may release chemotactic factors that amplify the response secondarily. Colchicine blocked the acute response to intrapleural asbestos by a mechanism yet to be understood.

Secretion of chemotaxins by guinea pig lung macrophages. I. The spectrum of inflammatory cell responses.

Chemotactic activity for neutrophils, macrophages, and lymphocytes was detected in the medium of guinea pig lung macrophage cultures. For the phagocytic cells, activity was greatest for neutrophils and least for lung macrophages. Activity for peritoneal macrophages was intermediate. Although the response of lymphocytes could not be directly compared to that of phagocytes, migration of lymphocytes toward culture supernatant greatly exceeded migration toward fresh medium. A phagocytic stimulus enhanced the activity for neutrophils in medium from cultures incubated for 2 hr. By 24 hr of incubation, however, activity in medium of stimulated and unstimulated cultures was similar. Since the increase in activity occurring over an 18 hr culture incubation period was not associated with loss of cell viability, it appears that release of chemotactic activity is a specific physiologic event. The finding that cultured lung macrophages secrete potent chemoattractants for neutrophils, macrophages, and lymphocytes in vi...

Proteoglycans, proteases, chemotaxis, and aggregation of inflammatory, cells.

A trypsin-like protease activity present in rabbit alveolar macrophages was shown to be activated by hyaluronic acid (HA) at 10(-9) M, but not by other proteoglycans. This activation of proteases by HA was accompanied by aggregation of alveolar macrophages and by the release of chemotactic activity. This chemotactic activity was destroyed by proteases and was less active for polymorphonuclear leukocytes than for alveolar macrophages. These effects of HA appear to be sepcific for alveolar macrophages, because no similar effects of HA on human polymorphonuclear leukocytes or lymphocytes could be observed. These observations suggest that HA, which is secreted into airways, plays a specific role in the macrophage-mediated chronic inflammatory processes in the distal lung.

Mediator release from basophil granulocytes in chronic myelogenous leukemia: Demonstration of the eosinophil chemotactic activity of histamine

Mediator release from the leukocytes of two patients with chronic myelogenous leukemia and basophilia was studied using rabbit antihuman IgE antibody. The release of histamine, slow reacting substance of anaphylaxis (SRS-A), platelet activating factor (PAF), chemotactic activity for neutrophils and eosinophils, and an inhibitor of eosinophil migration was observed. However, the release of SRS-A from the basophils of one patient and the release of chemotactic activity from both patients displayed unusual properties. During acceleration of the disease process, the basophils of one patient released maximal SRS-A activity at progressively lower concentrations of anti-IgE. Both patients released a high molecular weight factor (M.W. >20,000) which enhanced the migration of neutrophils and eosinophils and a low molecular weight chemotactic factor (M.W. < 500) which selectively attracted eosinophils. A double peak of eosinophil chemotactic activity was routinely observed for the low molecular weight factor; this was shown to represent the eosinophil chemotactic activity of histamine with relative inhibition of migration at the histamine peak. There was little release of the tetrapeptides, ECF-A, in these patients which facilitated demonstration of this eosinophilotactic activity of histamine. These results suggest that the eosinophil chemotactic activity observed in acute allergic reactions is the net effect of the release of multiple chemotactic factors.

Polymorphonuclear leukocyte motility in vitro. V. Release of chemotactic activity following phagocytosis of calcium pyrophosphate crystals, diamond dust, and urate crystals.

Abstract Phagocytosis of crystalline calcium pyrophosphate dihydrate (CPPD), but not amorphous calcium pyrophosphate, by human polymorphonuclear leukocytes (PMN) resulted in the generation of nondialyzable chemotactic activity. CPPD crystals did not generate as much chemotactic activity from PMN as urate crystals; however, this may have been due to a lower level of PMN phagocytosis of CPPD crystals. Colchicine caused a 70 to 100 per cent reduction in chemotactic activity in 710 urate crystal experiments and 49 to 70 per cent reduction in the remaining 3 experiments. By contrast, in no experiment with CPPD crystals was there greater than 70 per cent inhibition and in 4 experiments there was only 0 to 11 per cent inhibition. Diamond dust crystals were phagocytized by PMN, but no chemotactic activity was found. Release of chemotactic activity, therefore, is not dependent solely on crystal phagocytosis. Diamond dust crystals injected into canine joints did not induce an acute inflammatory response.