Gail L. Griffin

Chemotaxis of monocytes and neutrophils to platelet-derived growth factor.

The platelet-derived growth factor (PDGF) is shown to be chemotactic for monocytes and neutrophils. Maximum monocyte chemotaxis to PDGF is fully equal to that achieved with C5a and occurs at congruent to 20 ng/ml (congruent to 0.7 nM). Maximum neutrophil chemotaxis is congruent to 60% that achieved with C5A but occurs at congruent to 1 ng/ml (congruent to 32 pM). The chemotactic activity of PDGF is blocked by specific antisera to PDGF and by protamine sulfate, a competitive inhibitor of PDGF binding to cell surfaces. In contrast to PDGF, epidermal growth factor shows no chemotactic activity for inflammatory cells at 0.5-100 ng/ml. The high level of chemotactic activity of PDGF suggests that in addition to its role as a mitogen for smooth muscle cells and fibroblasts, PDGF may be involved in attracting inflammatory cells to sites of platelet release.

Chemotactic activity of elastin-derived peptides.

Elastin-derived peptides, produced by digesting human aortic elastin and bovine ligament elastin with human neutrophil elastase, were tested for chemotactic activity. At 100 micrograms protein/ml, elastin digests were nearly as active for monocytes as saturating amounts of complement-derived chemotactic activity. Neutrophils and alveolar macrophages showed less response to elastin peptidces than did monocytes. Fractionation of the digests by gel filtration chromatography disclosed that maximal chemotactic activity eluted in fractions corresponding to 14,000-20,000 mol wt containing most of the desmosine cross-links in the digests. Whole human serum and rabbit anti-elastin immunoglobulin inhibited the chemotactic activity. Purified desmosine also showed chemotactic activity for monocytes, maximal at 10 nM. These findings suggest that elastin-degradation products enriched in cross-linking regions recruit inflammatory cells in vivo and that elastin proteolysis, characteristic of emphysema, may be a signal for recruitment of mononuclear phagocytes into the lungs.

Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B.

Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents.

Chemotactic responses of fibroblasts to tropoelastin and elastin-derived peptides.

Fibroblasts are known to have chemotactic responses to two components of the extracellular matrix, collagen and fibronectin. To extend these observations to other extracellular connective tissue macromolecules and their proteolytic fragments, fibroblasts from adult human skin and from late-gestation (270 d), fetal bovine ligaments were studied for chemotactic responsiveness to tropoelastin and elastin-derived peptides. Bovine ligament tropoelastin and elastin-derived peptides, generated from either human aortic elastin with human neutrophil elastase or from bovine ligament elastin with pancreatic elastase, elicited chemotactic responses that were maximal at 0.2 micrograms/ml (3 X 10(-9) M) and 0.5-2.0 micrograms protein/ml, respectively. Fractionation of the elastin-derived peptides by gel filtration (Bio-Gel P-10) indicated that comparable levels of chemotactic activity were present in all fractions, and amino acid analysis of the fractions showed no relationship between chemotactic activity and desmosine concentration. Taken in conjunction with the observations on tropoelastin, it appears that fibroblast chemotaxis to elastin components does not involve the cross-links of elastin. These results demonstrate that the influences of the connective tissue matrix upon fibroblast migration might include elastin precursors and fragments of elastin.

Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin.

Entactin is an integral component of basement membranes that plays a major role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Because neutrophil (PMN) interactions with entactin have not been examined, we investigated the ability of natural and recombinant entactin to mediate PMN adhesion and chemotaxis. With both forms of entactin, we observed that entactin-coated surfaces promoted PMN adhesion and that entactin stimulated PMN chemotaxis. The increase in adhesion to entactin over control was two to threefold whereas the chemotactic response to 15 ng/ml (1 x 10(-10) M) entactin was equivalent to the chemotactic response elicited with 1 x 10(-8) M formyl-methionyl-leucyl-phenylalanine (fMLP). HL-60 cells, after differentiation with dimethylsulfoxide, also demonstrated adhesion and chemotaxis to entactin. A synthetic peptide of the Arg-Gly-Asp (RGD) domain in entactin, SIGFRGDGQTC (S-RGD), mediated PMN adhesion and chemotaxis, and preexposure of PMN to S-RGD blocked PMN adhesion and chemotaxis induced by entactin without diminishing the adhesive and chemotactic activities of fMLP. In contrast, preexposure to peptides SIGFRGEGQTCA or SIGFKGDGQTCA had no effect. The findings with synthetic peptides were confirmed with a recombinant entactin mutant in which aspartic acid at residue 674 was replaced with glutamic acid, thus converting the RGD sequence of entactin to RGE. RGE-entactin was neither adhesive nor chemotactic for neutrophils. Monoclonal antibodies to the leukocyte response integrin (LRI) and the integrin-associated protein blocked entactin-mediated adhesion and chemotaxis whereas monoclonal antibodies to beta 1 and beta 2 integrins had no effect and PMN from an individual with leukocyte-adhesion deficiency adhered normally to entactin-coated surfaces. These data demonstrate that entactin mediates biologically and pathologically important functions of PMN through its RGD domain and that LRI, which has been shown previously to mediate RGD-stimulated phagocytosis, is also capable of mediating RGD-stimulated PMN adhesion and chemotaxis.

The serpin-enzyme complex (SEC) receptor mediates the neutrophil chemotactic effect of alpha-1 antitrypsin-elastase complexes and amyloid-beta peptide.

The serpin-enzyme complex (SEC) receptor mediates catabolism of alpha 1-antitrypsin (alpha 1-AT)-elastase complexes and increases in synthesis of alpha 1-AT in cell culture. The SEC receptor recognizes a pentapeptide domain on alpha 1-AT-elastase complexes (alpha 1-AT 370-374), and the same domain in several other serpins, amyloid-beta peptide, substance P, and other tachykinins. Thus, it has also been implicated in the biological properties of these ligands, including the neurotoxic effect of amyloid-beta peptide. In this study, we examined the possibility that the SEC receptor mediates the previously described neutrophil chemotactic activity of alpha 1-AT-elastase complexes, and whether the other ligands for the SEC receptor have neutrophil chemotactic activity. The results show that 125I-peptide 105Y (based on alpha 1-AT 359-374) binds specifically and saturably to human neutrophils, and the characteristics of this binding are almost identical to that of monocytes and hepatoma-derived hepatocytes. Peptide 105Y and amyloid-beta peptide mediate chemotaxis for neutrophils with maximal stimulation at 1-10 nM. Mutant or deleted forms of peptide 105Y, which do not bind to the SEC receptor, have no effect. The neutrophil chemotactic effect of alpha 1-AT-elastase complexes is blocked by antiserum to peptide 105Y and by antiserum to the SEC receptor, but not by control antiserum. Preincubation of neutrophils with peptide 105Y or substance P completely blocks the chemotactic activity of amyloid-beta peptide, but not that of FMLP. These results, therefore, indicate that the SEC receptor can be modulated by homologous desensitization and raise the possibility that pharmacological manipulation of this receptor will modify the local tissue response to inflammation/injury and the neuropathologic reaction of Alzheimers disease.

PDGF AA homodimers are potent chemoattractants for fibroblasts and neutrophils, and for monocytes activated by lymphocytes or cytokines.

The A-chain homodimers of the platelet-derived growth factor (PDGF AA) are widely expressed in normal and transformed cells. The mitogenic properties of PDGF AA are well established; however, the chemotactic potential of PDGF AA remains controversial. We now show that PDGF AA is a strong chemoattractant for human monocytes, granulocytes, and fetal bovine ligament fibroblasts. However, highly purified (greater than 98%) monocytes require the addition of lymphocytes or IL-1 for chemotactic responsiveness to PDGF AA but not for full chemotactic activity with formyl-methionyl-leucyl-phenylalanine (fMLP) or C5a. These results indicate that PDGF AA is a potent chemoattractant. These results also indicate that monocytes require activation either by lymphocytes or exogenous cytokines in order to respond chemotactically to PDGF AA but not to fMLP or C5a and suggest roles of the lymphocyte and cytokine in the chemotactic response of the monocyte to PDGF AA in vivo.

Distal Airways in Mice Exposed to Cigarette Smoke Nrf2-Regulated Genes Are Increased in Clara Cells

Cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Terminal bronchioles are critical zones in the pathophysiology of COPD, but little is known about the cellular and molecular changes that occur in cells lining terminal bronchioles in response to CS. We subjected C57BL/6 mice to CS (6 d/wk, up to 6 mo), looked for morphologic changes lining the terminal bronchioles, and used laser capture microdissection to selectively isolate cells in terminal bronchioles to study gene expression. Morphologic and immunohistochemical analyses showed that Clara cell predominance remained despite 6 months of CS exposure. Since Clara cells have a role in protection against oxidative stress, we focused on the expression of antioxidant/detoxification genes using microarray analysis. Of the 35 antioxidant/detoxification genes with at least 2.5-fold increased expression in response to 6 months of CS exposure, 21 were NF-E2-related factor 2 (Nrf2)-regulated genes. Among these were cytochrome P450 1b1, glutathione reductase, thioredoxin reductase, and members of the glutathione S-transferase family, as well as Nrf2 itself. In vitro studies using immortalized murine Clara cells (C22) showed that CS induced the stabilization and nuclear translocation of Nrf2, which correlated with the induction of antioxidant and detoxification genes. Furthermore, decreasing Nrf2 expression by siRNA resulted in a corresponding decrease in CS-induced expression of several antioxidant and detoxification genes by C22 cells. These data suggest that the protective response by Clara cells to CS exposure is predominantly regulated by the transcription factor Nrf2.

Domain-specific Interactions between Entactin and Neutrophil Integrins: G2 DOMAIN LIGATION OF INTEGRIN α3β1 AND E DOMAIN LIGATION OF THE LEUKOCYTE RESPONSE INTEGRIN SIGNAL FOR DIFFERENT RESPONSES

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors. We show that the E domain, which contains the single RGD sequence of entactin, is sufficient for ligation of the β3-like integrin, leukocyte response integrin, and signaling for chemotaxis. Moreover, the G2 domain signals for stimulation of Fc receptor-mediated phagocytosis via ligation of α3β1. This receptor-ligand interaction was revealed only after stimulation of neutrophil by immune complexes or phorbol esters. Interestingly, the E domain does not enhance phagocytosis, and the G2 domain is not chemotactic. Furthermore, cleavage of entactin with the matrix metalloproteinase, matrilysin, liberates peptides that retain E domain-mediated chemotaxis and G2 domain-mediated enhancement of phagocytosis. These studies indicate that multiple domains of entactin have the ability to ligate individual integrins expressed by neutrophils and to activate distinct functions.

The carboxyl-terminal tridecapeptide of platelet factor 4 is a potent chemotactic agent for monocytes

Abstract The carboxyl-terminal tridecapeptide of platelet factor 4, Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser, was synthesized and found to have chemotactic activity towards monocytes at concentrations 1 30 that of the parent molecule, while eliciting the migration of cells comparable in number to that seen using the parent and the control C5a. In an α-helical conformation the tridecapeptide would be amphiphilic, making it a good candidate for binding to membrane surfaces. Indeed, addition of sodium dodecyl sulfate, an amphiphile, induces an increase in the peptides α-helicity, as shown by the circular dichroism spectrum. Pro-Leu-Tyr methyl ester was found to be inactive, indicating that the N-terminal region is not the recognition site of the tridecapeptide. These results suggest that the tridecapeptide incorporates the active site of human platelet factor 4 for monocyte chemotaxis.