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1.
Immunopharmacology ; 48(1): 17-26, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10822085

ABSTRACT

We investigated chemokine responses of human airway epithelial cells to transforming growth factor (TGF)-beta alone and in combination with tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. TGF-beta selectively induced production of granulocyte-macrophage colony stimulating factor (GM-CSF) without significant coordinate expression of IL-8 or RANTES. TNF-alpha induced expression of both IL-8 and GM-CSF, without detectable production of RANTES. TGF-beta synergistically enhanced GM-CSF production with TNF-alpha, but suppressed production and release of IL-8. IFN-gamma induced RANTES production and release; TGF-beta synergistically enhanced RANTES release induced by IFN-gamma, but had no effect on RANTES mRNA production. Taken together, these data demonstrate that TGF-beta may play a pivotal role in the responsiveness of airway epithelial cells to chemotactic cytokines, by selectively enhancing GM-CSF and RANTES production while suppressing IL-8 production. This profile of chemokine responses promoted by TGF-beta would favor eosinophil, lymphocyte and monocyte recruitment, hallmarks of chronic and allergic inflammation, over neutrophil sequestration.


Subject(s)
Bronchi/drug effects , Chemokines/biosynthesis , Transforming Growth Factor beta/pharmacology , Bronchi/immunology , Cells, Cultured , Chemokine CCL5/biosynthesis , Dose-Response Relationship, Drug , Drug Synergism , Epithelial Cells/drug effects , Epithelial Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Humans , Interferon-gamma/pharmacology , Interleukin-8/biosynthesis , RNA, Messenger/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology
2.
Immunopharmacology ; 46(3): 209-22, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10741901

ABSTRACT

Effects of the anaphylatoxins C3a and C5a on eosinophil and neutrophil adhesion to HUVEC and to primary culture human bronchial epithelial cells (HBEC) were investigated. Activities on both leukocytes and on structural cells were examined. C3a upregulated beta2 integrin expression and caused shedding of L-selectin on eosinophils, but had no effect on neutrophil adhesion molecule expression. C5a upregulated beta2 integrins and caused shedding of L-selectin on both eosinophils and neutrophils. The potency of C5a was equivalent on both cell types; however, the magnitude of the changes in each of these adhesion molecules was significantly greater in neutrophils than eosinophils. Neither C3a nor C5a altered expression of ICAM-1, VCAM-1, E-selectin or P-selectin on either HUVEC or HBEC. C5a induced adhesion of both neutrophils and eosinophils to unstimulated HUVEC or HBEC, and adhesion was further enhanced when HUVEC and HBEC were "primed" with TNF-alpha and IFN-gamma, respectively. C3a failed to enhance adhesion of either eosinophils or neutrophils to unprimed HUVEC or HBEC, and enhanced only eosinophil adhesion to cytokine-primed HUVEC or HBEC. Similar to C3a, C3a(desArg) and a C3a-analog peptide E7 also enhanced eosinophil adhesion only to cytokine-primed HUVEC and HBEC. These results support the traditional view of anaphylatoxins as leukocyte-specific mediators. The specificity of C3a for eosinophils implicates this molecule as a potential participant in allergic inflammation. The pro-adhesive effects of C3a(desArg) suggest that this molecule, previously characterized as a spasmogenically inactive derivative of C3a, may also alter leukocyte dynamics and migration. Finally, activation of endothelium may represent an important control mechanism for C3a-mediated adhesion preventing unchecked eosinophil adhesion to uninflamed systemic vasculature.


Subject(s)
Complement C3a/pharmacology , Complement C5a/pharmacology , Endothelium, Vascular/cytology , Eosinophils/physiology , Neutrophils/physiology , CD18 Antigens/analysis , Cell Adhesion/drug effects , Cells, Cultured , Epithelial Cells/cytology , Humans , Intercellular Adhesion Molecule-1/analysis , L-Selectin/analysis , Vascular Cell Adhesion Molecule-1/analysis
4.
Allergy Asthma Proc ; 20(4): 243-53, 1999.
Article in English | MEDLINE | ID: mdl-10476324

ABSTRACT

Production of GM-CSF by epithelial cells has been implicated in eosinophil survival within the airways, although GM-CSF promotes neutrophil and monocyte survival as well. Using primary cultures of human airway epithelial cells, we undertook a comprehensive examination of factors that enhance eosinophil survival or apoptosis. Unstimulated epithelial cells were compared to epithelial cells stimulated with TNF-alpha in the presence or absence of dexamethasone. A striking increase in survival was observed when peripheral blood eosinophils were cultured with supernatants derived from unstimulated and TNF-alpha-stimulated epithelial cells. Cultured epithelial cells were examined for transcripts of cytokines shown to enhance eosinophil survival (GM-CSF, IL-3, IL-5, IL-13, and IFN-gamma), and transcripts for cytokines promoting apoptosis (IL-10 and TGF-beta). GM-CSF transcripts, but not the other cytokines, were present in unstimulated epithelial cells, and levels were increased with TNF-alpha stimulation. TNF-alpha stimulation increased the levels of GM-CSF and PGE2 in epithelial cell supernatants and dexamethasone suppressed the TNF-alpha induced increases. The survival effects of the TNF-alpha-stimulated supernatants were effectively blocked by neutralizing antibodies to GM-CSF or by dexamethasone treatment of epithelial cells. Selectivity of GM-CSF for eosinophil versus neutrophil survival was demonstrated and suggests that epithelial cell regulation of GM-CSF and PGE2 contribute to eosinophil survival in vitro and may contribute to eosinophil accumulation in allergic disease.


Subject(s)
Dinoprostone/biosynthesis , Eosinophils/physiology , Glucocorticoids/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Respiratory System/immunology , Tumor Necrosis Factor-alpha/physiology , Apoptosis , Cell Survival , Cells, Cultured , Dexamethasone/pharmacology , Drug Interactions , Enzyme-Linked Immunosorbent Assay , Eosinophils/drug effects , Epithelial Cells/physiology , Glucocorticoids/pharmacology , Humans , Reference Values , Respiratory System/cytology , Sensitivity and Specificity , Tumor Necrosis Factor-alpha/pharmacology
5.
Am J Respir Cell Mol Biol ; 21(3): 418-27, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10460760

ABSTRACT

Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN-gamma. Maximal adhesion of Eos and PMN required pretreatment of HBECs with either TNF-alpha or IFN-gamma in addition to leukocyte activation. Adherence of Eos to unstimulated HBECs was mediated through both beta(1) and beta(2) integrins, whereas adhesion of Eos to activated HBECs was dominated by beta(2) integrins. Adhesion of both Eos and PMN was inhibited by treatment of HBECs with blocking antibodies to ICAM-1. Differential utilization of beta(1) and beta(2) integrins by Eos, depending on the activation state of the epithelium, is a novel finding and may affect activation and/or recruitment of Eos in airway tissue. Mechanisms of adhesion of HBECs to Eos and PMN, as evidenced by the different responsiveness of the two latter types of cells to IL-8 and IL-5, may account for a prevalence of Eos over PMN in certain airway diseases.


Subject(s)
Cell Adhesion/physiology , Eosinophils/physiology , Neutrophils/physiology , Bronchi/metabolism , CD18 Antigens/metabolism , Cells, Cultured , Cytokines/pharmacology , Dose-Response Relationship, Immunologic , Eosinophils/metabolism , Epithelial Cells/metabolism , Flow Cytometry , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Integrins/immunology , Integrins/physiology , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Interleukin-8/metabolism , Neutrophils/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Trachea/metabolism , Vascular Cell Adhesion Molecule-1/immunology , Vascular Cell Adhesion Molecule-1/metabolism
6.
Am J Respir Cell Mol Biol ; 21(2): 259-67, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10423410

ABSTRACT

We examined the potential of several epithelial-derived factors to enhance neutrophil activation and survival. Neutrophils incubated in the presence of supernatants from nasal-derived primary epithelial cultures had significantly increased survival compared with neutrophils cultured in media alone. Of the cytokines reported to enhance neutrophil survival, transcripts for interleukin (IL)-1alpha, IL-1beta, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF) (but not interferon-gamma or granulocyte colony-stimulating factor [G-CSF]) were detected by ribonuclease protection assay in basal and tumor necrosis factor (TNF)-alpha- stimulated epithelial cells. Of the eicosanoid products that enhance neutrophil survival, platelet-activating factor and leukotriene B(4) were not detected in the supernatants, whereas prostaglandin E(2) (PGE(2)) was produced in modest amounts. The levels of IL-6, GM-CSF, and PGE(2) in epithelial supernatants were significantly increased after transient TNF-alpha stimulation. This induction was suppressed if dexamethasone (Dex) was added during TNF-alpha stimulation. Only IL-6, GM-CSF, and PGE(2) promoted neutrophil survival over the range of concentrations detected in the supernatants, and a combination of neutralizing antibodies to GM-CSF and IL-6 completely inhibited the enhanced neutrophil survival in epithelial supernatants. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and morphologic scoring of apoptotic neutrophils confirmed that epithelial supernatants, as well as purified IL-6, GM-CSF, and PGE(2) all delayed neutrophil apoptosis. Finally, the effects of Dex on neutrophil survival and on epithelial cytokine production were investigated. Dex independently prolonged neutrophil survival but suppressed epithelial production of survival-enhancing factors in a dose-dependent manner. The net effect of Dex appeared to favor neutrophil survival.


Subject(s)
Glucocorticoids/physiology , Neutrophils/physiology , Tumor Necrosis Factor-alpha/physiology , Apoptosis , Cell Survival , Cells, Cultured , Culture Media, Conditioned/metabolism , Dinoprostone/metabolism , Epithelial Cells/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , In Situ Nick-End Labeling , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/metabolism , Interleukin-6/metabolism , Sialoglycoproteins/metabolism , Time Factors
7.
J Immunol ; 162(2): 1127-36, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9916743

ABSTRACT

The comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1beta-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in alphaMbeta2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of alphaMbeta2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-alpha4 and anti-beta2 integrin mAbs. In vitro, both C3a (but not C3a(desArg)) and C5a (including C5a(desArg))-dependent transmigration of eosinophils across IL-1beta-stimulated endothelial monolayer was mediated by alpha4beta1 and alphaMbeta2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.


Subject(s)
Cell Movement/immunology , Complement C3a/physiology , Complement C5a/physiology , Endothelium, Vascular/immunology , Eosinophils/immunology , Venules/immunology , Animals , Antibodies, Monoclonal/pharmacology , CD18 Antigens/immunology , Cell Adhesion/immunology , Cell Adhesion Molecules/biosynthesis , Cell Movement/drug effects , Complement C3a/administration & dosage , Complement C5a/administration & dosage , Endothelium, Vascular/cytology , Hemodynamics , Humans , Infusion Pumps , Integrin alpha4beta1 , Integrins/immunology , Interleukin-1/pharmacology , Lysine Carboxypeptidase/pharmacology , Mesentery/blood supply , Neutrophils/immunology , Rabbits , Receptors, Lymphocyte Homing/immunology , Venules/cytology , Venules/physiology
8.
J Immunol ; 162(1): 453-9, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-9886419

ABSTRACT

The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3a(desArg) on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1beta. C3a or C3a(desArg) alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1beta, both C3a and C3a(desArg) were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1beta-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3a(desArg) enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3a(desArg), indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3a(desArg) on induction of NF-kappaB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-kappaB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro.


Subject(s)
Complement C3a/analogs & derivatives , Complement C3a/physiology , Interleukin-6/biosynthesis , Leukocytes, Mononuclear/metabolism , Amino Acid Sequence , Cells, Cultured , Dinoprostone/antagonists & inhibitors , Dinoprostone/biosynthesis , Humans , Indomethacin/pharmacology , Interleukin-1/pharmacology , Interleukin-6/blood , Interleukin-6/genetics , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/pharmacology , Molecular Sequence Data , NF-kappa B/biosynthesis , Pertussis Toxin , RNA, Messenger/metabolism , Transcription Factor AP-1/biosynthesis , Virulence Factors, Bordetella/pharmacology
9.
Immunopharmacology ; 39(3): 243-53, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9754910

ABSTRACT

A potential mechanism for the increased sensitivity of inflamed tissues to bradykinin is the upregulation of bradykinin receptor expression. We report that recombinant human IFNgamma stimulated a concentration-dependent increase in cell surface bradykinin receptor expression in intact T24 human epithelial-like cells, determined by radioligand binding analysis. Analysis of specific [3H]-bradykinin binding revealed that IFNgamma-treated cells had a two- to threefold increase in bradykinin receptor number compared to the controls with no effect on receptor affinity. The ability of IFNgamma to stimulate increased bradykinin receptor expression was abrogated by treatment with either the transcription inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. IFNgamma enhanced steady-state human B2 bradykinin receptor mRNA expression in the T24 cells in a dose-dependent manner. B2 bradykinin receptor mRNA expression was increased as early as 1 h following IFNgamma stimulation, and continued to accumulate for 24 h. Bradykinin-stimulated intracellular calcium mobilization was also increased in IFNgamma-treated T24 cells compared to controls. The ability of IFNgamma to upregulate B2 bradykinin receptors in primary epithelial cells was demonstrated using cultured human airway epithelial cells. These observations suggest that increasing IFNgamma levels during inflammation may upregulate the expression of B2 bradykinin receptors, leading to increased sensitivity to bradykinin.


Subject(s)
Interferon-gamma/pharmacology , RNA, Messenger/biosynthesis , Receptors, Bradykinin/biosynthesis , Bradykinin/metabolism , Bronchi/drug effects , Bronchi/metabolism , Bronchi/ultrastructure , Calcium/metabolism , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Humans , Kinetics , RNA, Messenger/metabolism , Receptor, Bradykinin B2 , Recombinant Proteins , Stimulation, Chemical , Trachea/drug effects , Trachea/metabolism , Trachea/ultrastructure , Up-Regulation
10.
J Immunol ; 160(6): 3038-45, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9510209

ABSTRACT

Recent evidence suggests a novel role of bradykinin (BK) in stimulating gene transcription. This study examined the effect of BK on nuclear factor kappaB (NF-kappaB) activation and IL-1beta synthesis in human epithelial cells. Stimulation of A549 cells and primary bronchial epithelial cells with BK rapidly activated NF-kappaB. BK also increased the level of secreted immunoreactive IL-1beta in A549 culture supernatants, an effect that was blocked by actinomycin D and the B2 BK receptor antagonist HOE-140. The role of NF-kappaB activation in BK-induced IL-1beta synthesis was demonstrated by the ability of BK to stimulate increased chloramphenicol acetyltransferase (CAT) activity in A549 cells transfected with a reporter plasmid containing three kappaB enhancers from the IL-1beta gene, while deletion of the kappaB enhancer sequences eliminated BK-stimulated CAT activity. C3 transferase exoenzyme, an inhibitor of Rho, abolished BK-induced NF-kappaB activation at 10 microg/ml and significantly inhibited BK-stimulated IL-1beta synthesis at 5 microg/ml. A dominant-negative form of RhoA (T19N) inhibited BK-stimulated reporter gene expression in a dose-dependent and kappaB-dependent manner. Cotransfection of A549 cells with an expression vector encoding a constitutively active form of RhoA (Q63L) along with the IL-1beta promoter-CAT reporter plasmid resulted in a marked increase in NF-kappaB activity compared with transfection with the IL-1beta promoter-CAT reporter plasmid alone. These results demonstrate that BK stimulates NF-kappaB activation and IL-1beta synthesis in A549 cells, and that RhoA is both necessary and sufficient to mediate this effect.


Subject(s)
Bradykinin/pharmacology , GTP-Binding Proteins/physiology , Interleukin-1/genetics , NF-kappa B/metabolism , Bronchi/metabolism , Cells, Cultured , Chloramphenicol O-Acetyltransferase/metabolism , DNA/metabolism , Epithelial Cells/metabolism , Humans , Interleukin-1/biosynthesis , rhoA GTP-Binding Protein
11.
J Leukoc Biol ; 63(1): 75-82, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9469475

ABSTRACT

Circulating serine proteinase inhibitors (serpins) regulate a number of proteinases that participate in the inflammatory process. In this study, we investigated possible modulatory effects of serpins on neutrophil adhesion. Antichymotrypsin (ACT), alpha1-protease inhibitor (alpha1-PI), and LEX032, a recombinant hybrid of ACT and alpha1-PI were shown to inhibit neutrophil adhesion to fibronectin (FN)-coated surfaces and, to a lesser extent, adhesion to other extracellular matrix proteins. The inhibitory effect of serpins on neutrophil adhesion to FN was found to be related to inhibition of FN proteolysis based on the following observations: (1) elastase treatment of FN-coated plates, but not of neutrophils, resulted in enhanced neutrophil adhesion; and (2) serpins inhibited FN proteolysis by neutrophil proteases. Serpins also inhibited neutrophil spreading, as well as shedding of neutrophil CD43, but not L-selectin, CD18, or CD29. We conclude that serpins modulate neutrophil adhesion both by inhibiting proteolytic processing of extracellular matrix proteins and proteolytic shedding of CD43.


Subject(s)
Antigens, CD , Chymotrypsin/antagonists & inhibitors , Neutrophil Activation , Neutrophils/physiology , Serine Proteinase Inhibitors/pharmacology , alpha 1-Antitrypsin/physiology , Cell Adhesion , Cell Adhesion Molecules/metabolism , Extracellular Matrix Proteins/metabolism , Eyeglasses , Fibronectins/metabolism , Humans , Leukosialin , Neutrophils/cytology , Recombinant Proteins , Sialoglycoproteins/metabolism
12.
Infect Immun ; 64(6): 1984-91, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8675297

ABSTRACT

The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils. Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsullonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at both potential Lys-gingipain sites (i.e., between residues 17 and 18 [K-D] and 28 and 29 [K-T]) and at two chymotrypsin sites (between residues 14 and 15 [Y-D] and 20 and 21 [L-D]), respectively. These studies suggest that P. gingivalis contains at least two enzymes capable of cleaving the C5aR, Lys-gingipain and a second nontryptic serine proteinase that is distinct from either Arg- or Lys-gingipain.


Subject(s)
Antigens, CD/metabolism , Endopeptidases/pharmacology , Porphyromonas gingivalis/enzymology , Receptors, Complement/metabolism , Amino Acid Sequence , Animals , Calcium/metabolism , Chymotrypsin/pharmacology , Humans , Molecular Sequence Data , Neutrophils/enzymology , Rabbits , Receptor, Anaphylatoxin C5a , Trypsin/pharmacology
13.
Adv Exp Med Biol ; 389: 155-64, 1996.
Article in English | MEDLINE | ID: mdl-8861006

ABSTRACT

The anaerobic bacteria P. gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacteria and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Collagenases and cysteine proteinases (i.e., the gingipains) have been characterized as the predominant vesicular enzymes produced by this bacterium. It has been shown that an arginine-specific cysteine proteinase from P. gingivalis, called gingipain-1 or Arg-gingipain, can selectively cleave complement components C3 and C5. In the case of C5, cleavage by Arg-gingipain results in the generation of C5a, a potent chemotactic factor for PMNs. Since these bacterial proteinases are capable of generating pro-inflammatory factors at sites of infection, we examined the possibility that gingipains or other proteinases from this bacterium might attack or destroy cell surface proteins, such as receptor molecules. Using an affinity-purified rabbit antibody raised against residues 9-29 of the C5a receptor (i.e., C5aR; CD88), the signal transmitting element for the pro-inflammatory mediator C5a, we demonstrated that the mixture of proteinases in P. gingivalis vesicles cleaves the C5a receptor on human neutrophils. This vesicular proteinase activity did not require cysteine activation which indicates that proteinases other than the gingipains may be responsible for cleavage of the C5aR molecule. in addition, the purified Lys-gingipain, but not Arg-gingipain, also cleaved C5aR on the human neutrophils. The N-terminal region of CaR (residues 9-29, PDYGHYDDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin, but not by trypsin, despite the presence of potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of C5aR 9-29 peptide cleavage were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain. These studies suggest that the proteolytic activity in the bacterial vesicles that is responsible for cleaving C5aR is primarily a non-tryptic proteinase, distance from either Arg- or Lys-gingipain. Consequently, there appear to be additional proteinase(s) in the vesicles that attacks the cell surface molecule C5aR which are not the same (i.e., Arg- and Lys-gingipain) as were shown to generate pro-inflammatory activity from complement components C3 and C5. Evidence that the proteinases which attack the inflammatory precursor molecules (i.e., C3 and C5) exhibit different specificities than those that attack receptors to these bioactive complement products makes a particularly interesting story of how this bacteria avoids major host defense mechanisms. It is well known that generation of pro-inflammatory factors such as C3a and C5a at extra-vascular sites can promote edema, leukocyte recruitment and cellular activation responses that could lead to the release of toxic oxygen products and to phagocytosis of the bacteria. Destruction of receptors to these cellular activating factors generated by bacterial proteinases may eliminate the ability of these (i.e., complement-derived) and other mediators to carry out their anti-bacterial actions and thereby limit the host's defense mechanisms in responses to the infecting bacteria. The concept of anti-bacterial responses (i.e., oxygen radical generation and phagocytosis) being effectively eliminated at the injury site, by bacterial proteinases acting at the cellular receptor level, has not been studied in detail. In this case, the situation is particularly unusual because, once the bacterial gingipains generate potent plasma-derived inflammatory factors that can enhance edema and deliver essential nutrients to the bactgeria, other bacterial proteinases may destsroy their cellular receptors. These receptors transmit the signal activation mechanisms in the infiltrating cells that elicit bacterial killing.(ABSTRACT TRUNCATED)


Subject(s)
Antigens, CD/blood , Complement C5a , Cysteine Endopeptidases/blood , Endopeptidases/blood , Hemagglutinins/blood , Leukocytes/metabolism , Porphyromonas gingivalis/enzymology , Receptors, Complement/blood , Adhesins, Bacterial , Amino Acid Sequence , Cysteine Endopeptidases/isolation & purification , Endopeptidases/isolation & purification , Gingipain Cysteine Endopeptidases , Hemagglutinins/isolation & purification , Humans , Molecular Sequence Data , Receptor, Anaphylatoxin C5a , Signal Transduction/physiology
14.
Blood ; 85(10): 2900-9, 1995 May 15.
Article in English | MEDLINE | ID: mdl-7537984

ABSTRACT

We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF-alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors.


Subject(s)
CD18 Antigens/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion , Complement C5a/pharmacology , Integrins/metabolism , Leukocytosis/pathology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antibodies, Monoclonal , In Vitro Techniques , L-Selectin , Leukocytosis/chemically induced , Male , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Rabbits
15.
Immunopharmacology ; 28(1): 1-18, 1994.
Article in English | MEDLINE | ID: mdl-7928299

ABSTRACT

Homeostatic mechanisms controlling levels of circulating leukocytes have been an enigma in the field of hematology for decades. The short circulating half-life of PMNs relative to other leukocytic cell types, and their critical role as a front line of defense against infectious agents ascribes particular importance to this regulatory process. While strident advances have expanded our knowledge of how leukocytes develop and mature in the bone marrow, their regulation and mechanisms for transport into the circulation remain largely unexplained. The relatively recent availability of recombinant cytokines and other highly purified biologic mediators, as well as the development of monoclonal antibodies against specific leukocyte adhesion molecules have led to new insights and renewed interest in this dynamic process (Springer, 1990; Petrides and Dittmann, 1990). This article reviews recent advances in defining the cellular and molecular interactions involved in leukocyte recruitment by various mediators, and proposes conceptual models for regulation of circulating leukocyte levels.


Subject(s)
Chemotaxis, Leukocyte/physiology , Leukocytosis/immunology , Neutrophils/physiology , Animals , Bone Marrow Cells , Cell Adhesion/physiology , Chemotactic Factors/physiology , Cytokines/physiology , Humans
16.
J Trauma ; 36(5): 651-7, 1994 May.
Article in English | MEDLINE | ID: mdl-8189465

ABSTRACT

The aim of this study was to search for early inflammatory mediators in severely traumatized patients that could predict the occurrence of adult respiratory distress syndrome (ARDS). We measured sequential plasma levels of tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), complement fragment C3a, and endotoxin. In addition, we measured sequentially the values of hemodynamics, oxygen transport, and pulmonary function. The temporal patterns seen in the patients who developed ARDS were compared with those who did not. In the patients who developed ARDS, the first observed findings were low cardiac index (CI) and oxygen delivery (DO2) followed by progressive increases in IL-6, IL-8 and C3a levels, worsening of pulmonary function, and increases in hemodynamic values. The maximum values of IL-6, IL-8, and C3a occurred after the onset of ARDS. In the patients who did not develop ARDS, initial oxygen transport values were not low, the levels of IL-6, IL-8, and C3a decreased rapidly from their initial peaks, and there were no further increases in hemodynamic values. In both ARDS and nonARDS patients, no measurable quantities of TNF, IL-1, or endotoxin were found. We concluded that none of the mediators we measured reached their peaks before the onset of ARDS and none were found to be predictive of posttraumatic ARDS. However, these and other mediators may augment or intensify the development of ARDS.


Subject(s)
Cytokines/blood , Respiratory Distress Syndrome/physiopathology , Wounds and Injuries/physiopathology , Adult , Complement C3a/analysis , Hemodynamics , Humans , Interleukins/blood , Middle Aged , Oxygen Consumption , Prospective Studies , Respiratory Distress Syndrome/blood , Respiratory Distress Syndrome/etiology , Time Factors , Wounds and Injuries/blood , Wounds and Injuries/complications
17.
Crit Care Med ; 22(5): 768-76, 1994 May.
Article in English | MEDLINE | ID: mdl-8181284

ABSTRACT

OBJECTIVE: The adult respiratory distress syndrome (ARDS) is a frequent complication after severe accidental trauma. This study examines the hypothesis that increased systemic concentrations of proinflammatory cytokines, endotoxin, or complement fragments may predict the development of ARDS. DESIGN: Prospective, observational study. SETTING: Two Level I university trauma centers. PATIENTS: Fifteen severely injured patients (Injury Severity Score of > or = 25). INTERVENTIONS: Standard emergency department, operating room, and intensive care unit management. MEASUREMENTS AND MAIN RESULTS: Plasma samples were obtained at 4-hr intervals from the time of injury and were assayed for concentrations of endotoxin, tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, IL-8, and complement fragments C3a and C4a. Hemodynamic and oxygen metabolism variables also were measured at 4-hr intervals after injury. Seven patients developed ARDS and eight patients did not. The PaO2/FIO2 ratio was significantly decreased in the patients with ARDS compared with non-ARDS patients as early as 4 hrs postinjury, and remained significantly decreased throughout the initial 24 hrs after severe accidental injury. Plasma IL-8, IL-6, C3a, and C4a concentrations were markedly increased starting in the immediate postinjury period in both ARDS and non-ARDS patients, but no significant differences were found between the two groups until 16 hrs after injury when plasma IL-8, C3a, and C4a concentrations became significantly higher in the ARDS group. Neither the ARDS nor non-ARDS patients showed the presence of circulating IL-1 beta, TNF-alpha, or endotoxin at any postinjury time point. CONCLUSIONS: These results demonstrate that measurements of plasma concentrations of proinflammatory cytokines, endotoxin, or complement fragments are not helpful in predicting the development of ARDS after severe accidental injury.


Subject(s)
Complement C3a/analysis , Complement C4a/analysis , Craniocerebral Trauma/complications , Endotoxins/blood , Hemodynamics , Interleukin-1/blood , Interleukin-6/blood , Interleukin-8/blood , Peptide Fragments/blood , Respiratory Distress Syndrome/blood , Respiratory Distress Syndrome/epidemiology , Tumor Necrosis Factor-alpha/analysis , Adult , Blood Gas Analysis , Craniocerebral Trauma/classification , Enzyme-Linked Immunosorbent Assay , Female , Humans , Injury Severity Score , Interleukin-1beta , Male , Middle Aged , Oxygen Consumption , Predictive Value of Tests , Prognosis , Prospective Studies , Radioimmunoassay , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/physiopathology , Risk Factors , Survival Rate , Time Factors
18.
J Immunol ; 148(4): 1119-28, 1992 Feb 15.
Article in English | MEDLINE | ID: mdl-1310708

ABSTRACT

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.


Subject(s)
Bone Marrow Cells , Chemotactic Factors/pharmacology , Leukocytosis/etiology , Neutrophils/drug effects , Animals , Cells, Cultured , Complement C5a/pharmacology , Dactinomycin/pharmacology , Interleukin-8/pharmacology , Leukotriene B4/pharmacology , Male , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Platelet Activating Factor/pharmacology , Pyrazoles/pharmacology , Rabbits , Receptors, Formyl Peptide , Receptors, Immunologic/analysis
19.
Immunobiology ; 181(4-5): 379-87, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2129204

ABSTRACT

Hemorrhage has multiple effects on immunologic response, including alteration of B cell repertoires and T cell function. This study examined possible relationships between these two phenomena by determining the effects of T cells and T cell subsets transferred from hemorrhaged donors into normal, unhemorrhaged syngeneic recipients on B cell repertoires. Mice given total T or CD8+ cells from hemorrhaged animals then immunized with the bacterial polysaccharide antigen levan had a decreased percentage of plasma cells producing antibody to levan compared to that in mice given T or CD8+ cells from unhemorrhaged animals. These effects of post hemorrhage CD8+ cells were not seen after transfer into nu/nu mice, indicating that these cells did not directly affect B cell function, but rather required other T cell populations in order to alter the B cell repertoire. These results demonstrate that hemorrhage-induced alterations in bacterial antigen specific B cell repertoires may result from T and CD8+ cell mediated changes in T-B interactions.


Subject(s)
Hemorrhage/immunology , T-Lymphocyte Subsets/immunology , Animals , Antigens, Bacterial , Antigens, Differentiation, T-Lymphocyte , CD8 Antigens , Fructans/immunology , Immunization, Passive , Male , Mice , Mice, Inbred BALB C , Plasma Cells/immunology
20.
J Steroid Biochem ; 36(6): 659-66, 1990 Aug 28.
Article in English | MEDLINE | ID: mdl-2214783

ABSTRACT

Fluoxymesterone, an anabolic steroid, is metabolized in man primarily by 6 beta-hydroxylation, 4-ene-reduction, 3-keto-reduction, and 11-hydroxy-oxidation. These pathways of metabolism are suggested by the positive identification of 4 metabolites and the tentative identification of 3 other metabolites. Detection of the drug in urine is possible for at least 5 days after a single 10 mg oral dose to previously untreated adult males, by monitoring the presence of 2 metabolites, since the parent drug is not detectable more than 1 day after the dose.


Subject(s)
Fluoxymesterone/pharmacokinetics , Adult , Dose-Response Relationship, Drug , Fluoxymesterone/metabolism , Fluoxymesterone/urine , Gas Chromatography-Mass Spectrometry , Humans , Male , Models, Biological , Substance-Related Disorders/urine
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