- Email: [email protected]
Structure-function of chemokines
CYTOKINE,
544 I Abstracts
P3 October 2: Inflammation
Vol. 6, No. 5 (September
1994: 539-582)
and Cytokines
A3Q
A33 IL-18 AND LPS MEDIATE FIBROBLAST HYPERPLASIA THROUGH MODULATION OF THE PDGF RECEPTOR SYSTEM. J. Banner. P. Lindroos, P. Coin, A. Badgett and A. Osornio-Vargas National inst. of Environ. Health Sciences, NIH, Res. Triangle Pk., NC 27709 Platelet-derived growth factor (PDGF) isoforms are potent fibroblast mitogens. The significance of two PDGF receptor subtypes (a and 8) is not known. Bacterial lipopolysaccharide (LPS) induces PDGF and interleukin-1 8 (IL-18) synthesis by macrophages during inflammation. We postulated that LPS. either directly or via IL-18, could affect the fibroblast PDGF receptor system Normal lung fibroblasts possessed abundant mRNA for PDGF-8 receptor subtype (PDGF-R8) and a paucity of PDGF-a receptor (PDGF-Ra). In rnitogenesis and chemotaxis assays, these cells responded to PDGF-AB and PDGF-BB which bind PDGF-Ra and PDGF-R8, but not to PDGF-AA which binds only PDGF-Ra. Following IL-18 treatment, we observed a marked increase in PDGF-Ra 9ene expression that was msximal at 4 hrs. After 24 hrs, IL-18 (0.5 to 20 ns/ml) and LPS (3 to 100 “s/ml) stimulated a several-fold increase in [1251]PDGF-AA binding that was indicative of the cell-surface RDGF-Ha. [“H]Thymidine assays revealed that LPS and IL-1p pretreatment increased the mitogenic response of fibroblasts 2 to 3-fold to all three PDGF isoforms. Similarly, chemotaxis of fibroblasts to the PDGF isoforms was increased 2 to 3-fold following IL-18 treatment. These data suggest that, during inflammation, LPS or IL-18 render fibroblasts hyperresponsive to PDGF isoforms through upregulation of the PDGF-Fir.. We hypothesize that the PDGF-RCX ss~vss to mediate rapid proliferation 31fibroblasts during tissue repair and that the PDGF-RP, which is not afr-cted by these inflammatory mediators, is more Important to normal tissue maintainence.
IL-la PRODUCTION BY MONOCYTIC CELLS IS ENHANCED BY PLATELETS. Burton D. Clark, Ellen C. Donaldson, Koichi Aiura, Ronald G. Tompkins, Charles A. Dinarello. John F. Burke, and Jeffrey A. Gelfand. Dept. of.Meclicine, NEMC; Tufts Univ. School of Medicine: Mass. General Hospital; and Harvard Medical School, Boston, MA 02111 We have investigated the role of platelets with monocytes and their enhancement of cytokine production by the monocytic cell line, MonoMac 6. In cur studies, thrombin-activated platelets (P) were incubated with MonoMac 6 cells (M) at ratios of 0, 16, 80, 160, and 800 P/M with or without endotoxin (LPS) or heat-killed S. epidefmidis (SE). IL-la was quantitated by a specific and sensitive RIA subsequent to 4h and 8h incubations. The incubation of activated platelets with monocytic cells (16:l ratio) for 4h in the presence of LPS (M+P+LPS) at 50,100 and 500 pg/ml resulted in 3.6, 4.8, 4.9 fold increases in IL-la, respectively, compared to monocytes exposed to LPS without platelets (baseline, M+LPS). After 8h incubation, the overall percentage change from baseline decrease by approximately one-half, reflected by a corresponding decrease in baseline (M+LPS) cytokine production. Monocytes plus platelets incubated with S. epidermidis for 4 h (M+P+SE) showed a 3.5 fold increase over baseline (M+SE), and a 2 fold increase at 8h. There was a dose response for cytokine production with increased ratios of P:M. Overall, these results suggest that activated plateiets may magnify the cytokine response observed during both Gram-positive and Gram-negative infection.
A34
A31 INDUCIBLE EXPRESSION OF PTX3, A NEW MEMBER OF THE PENTRAXIN FAMILY, IN HUMAN MONONUCLEAR PHAGOCYTES. Barbara Bottazzi, VictorVidal Alles, Giuseppe Peri, JosCe Golay, Martin0 Introna and Albert0 Mantovani. Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy. PTX3 was recently cloned as an IL-l-inducible gene in endothelial cells, with structural similarities to pentraxins (C-reactive protein and Serum Amyloid P component). Aim of this study was to investigate the expression of PTX3 in human leukocytes populations. Human peripheral blood monocytes exposed to lipopolysaccaride or IL-lfi expressed significant levels of PTX3 messenger RNA (mRNA). TNFo. was a less effective inducer of PTX3, whereas IL-6, MCP-1, M-CSF, GM-CSF and IFN-7 were inactive. PTX3 transcript was undetectable in resting or stimulated polymorphonuclear cells, T or B lymphocytes and NK cells. PTX3 mRNA was also inducible in in vi&o monocytederived macrophages, tumor-associated macrophages and in the myelomonocytic cell lines HL60, U937 and THPl, but not in GFD8. Characterization of the binding properties of PTX3 is in progressusing a PTX3 specific antiserum, release of PTX3 protein was demonsrated for the first time in stimulated monocytes as well as in endothelial and fibroblastic cells. Thus, PTX3, unlike the classical pentraxins CRP and SAP, is expresed and released by cells of the monocyte-macrophage lineage exposed to inflammatory signals.
OF CHEMOKlNES, I.?, I.-H. Gongt, B. K. Rajaratbnam~, K.S. Kim%, and B.D. SykesP, TTbe Biomedical Research Centre and Biochemistry Department, UBC, Vancouver BC, V6T M; ‘Theodor-Kocher Institute, Uniwsity of Bern, Switzerland, CH 3CC0; ~Biochemishy Deparbnem, University of Alberta, Edmonton AB, T6G 2S2.
Recruitment of Mononuclear Cells to the Lung Using a Recombinant Adenovirus Expressing Murine RANTES. Todd A. Bra&k’, Zhou Xing’, Kevin Bacon’, Frank L. Graham’, Thomas J. Schall’, Carl D. Richards’, Ken Croitoru3 and Jack Gauldie’, Departments of Pathology’ and Medicine3, McMaster University, Hamilton, Canada L8N 325, DNAX Research Institute, Palo Alto, CA’. RANTES is a member of the C-C subfamily of chemokines that is reported to function as a chemoattractant for monocytes, eosinophils and CD4+ T cells. Using a recombinant human type 5 adenovirus (ADS) containing the murine RANTES cDNA (Ad5E3mRANTES), we initiated a study to characterize the biological functions of this cytokine in viva. Intratracheal administration with 2~10~ pfu of Ad5E3mRANTES or control virus targeted transgene expression to lung epithelial cells of Sprague-Dawley rats. Only Ad5E3mRANTES treated animals were found to have physiologic concentrations of RANTES cytokine detected in the bronchoalveolar lavage (BAL) fluids. Concurrent with the presence of RANTES cytokine were significant increases in the number of mononuclear cells. At 24h monocyte numbers were increased 50 fold in Ad5E3mRANTES treated rats. Histological examination of lung sections were consistent with the BAL data and indicates an active process of monocyte recruitment to the lung confirming the proinflammatory nature of RANTES in viva and its function as a monocyte chemoattractant. (Supported by MRC Canada)
QUANTIFICATION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST (IL-lra) GENE ALLELE-SPECIFIC TRANSCRIPTS IN KERATINOCYTES. F.E. Clay, J.K. Tarlow, MI. Cork and G.W. Duff. Section of Molecular Medicine, University of Sheffield, SlO 2JF, U.K. We have shown an allele of the IL-lra gene to be associated with the clinical outcome in several chronic inflammatory diseases. To test the functional correlate of this DNA variation, we measured mRNA from the two main alleles using a totally linked exonic marker. Allele 2 of this exonic polymorphism creates an Hpa II site, and is linked to the disease-related allele of IL-lra. RNA was isolated from keratinocytes and RT-PCR using a 5’ end-labelled primer was performed. The product was cut with Hpa II and analysed on a phosphorimager. The relative quantity of RNA from each allele was determined. In 9 of 12 heterozygotes, mRNA from allele 1 was significantly more abundant than from allele 2. Equal levels of mRNA were seen in 2 of 12 and 1 cell line showed more mRNA from allele 2 than from allele 1. This differential mRNA accumulation may be due to altered gene transcription or mRNA stability in the two alleles. Thus mRNA for this anti-inflammatory cytokine is less abundant in genotypes that have been associated with chronic inflammatory diseases.
S’IRUCTURE-FUNCTION Dewald*, M. Baggiolini*,
Functional and structural dialysis of interk&in-8 and other cbemokine analogs has provided insights into their recepmr interactions and stnxture-activity relatiombips. Interleukin-8. which has been shown to be a dim% was found to function as the monomeric form. Although struaml analysis revealed that MGSA is a dimcr in solution, it was found to dissociate to monomers at physiological concentrations. Analogs and CXC chemokine hybrids have shown &t &thin the IL-8 monomer, 4 smcbml motifs are immxtant: the N-terminal residues 4.5, and 6, termed the ELR motif; the N-tem&nJ ioop region that includes residues 1l-20; the 30-35 p turn; and tbe two disultide. bridges. It is hypothesized that the IL-8 protein, particularly the 30-35 p mm and the disultide bridges, provides a scaffold for orientation of the ELR motif, which is the primary receptor activation site. A Z-site receptor-binding model is proposed, similar to that suggested for CSa. In this model, receptor site 1 binds a secondary site of IL-8 comprising residues 11-20, whereas the primary receptor-binding site of IL-8, the ELR motif. binds a second, buried, receptor site. Results with MCP-1 analogs demons&ate the importance of the N-terminal region for hmction and, by analogy with IL-8, mmcated MCP-1 forms MCP-l-specific receptor antagonists. Thus, preliminary results with MCP-I suggest that the general principles established for the CXC chemckines will also apply to CC chemokines.
A32
A35 Tissue
Directed
544 I Abstracts
P3 October 2: Inflammation
Vol. 6, No. 5 (September
1994: 539-582)
and Cytokines
A3Q
A33 IL-18 AND LPS MEDIATE FIBROBLAST HYPERPLASIA THROUGH MODULATION OF THE PDGF RECEPTOR SYSTEM. J. Banner. P. Lindroos, P. Coin, A. Badgett and A. Osornio-Vargas National inst. of Environ. Health Sciences, NIH, Res. Triangle Pk., NC 27709 Platelet-derived growth factor (PDGF) isoforms are potent fibroblast mitogens. The significance of two PDGF receptor subtypes (a and 8) is not known. Bacterial lipopolysaccharide (LPS) induces PDGF and interleukin-1 8 (IL-18) synthesis by macrophages during inflammation. We postulated that LPS. either directly or via IL-18, could affect the fibroblast PDGF receptor system Normal lung fibroblasts possessed abundant mRNA for PDGF-8 receptor subtype (PDGF-R8) and a paucity of PDGF-a receptor (PDGF-Ra). In rnitogenesis and chemotaxis assays, these cells responded to PDGF-AB and PDGF-BB which bind PDGF-Ra and PDGF-R8, but not to PDGF-AA which binds only PDGF-Ra. Following IL-18 treatment, we observed a marked increase in PDGF-Ra 9ene expression that was msximal at 4 hrs. After 24 hrs, IL-18 (0.5 to 20 ns/ml) and LPS (3 to 100 “s/ml) stimulated a several-fold increase in [1251]PDGF-AA binding that was indicative of the cell-surface RDGF-Ha. [“H]Thymidine assays revealed that LPS and IL-1p pretreatment increased the mitogenic response of fibroblasts 2 to 3-fold to all three PDGF isoforms. Similarly, chemotaxis of fibroblasts to the PDGF isoforms was increased 2 to 3-fold following IL-18 treatment. These data suggest that, during inflammation, LPS or IL-18 render fibroblasts hyperresponsive to PDGF isoforms through upregulation of the PDGF-Fir.. We hypothesize that the PDGF-RCX ss~vss to mediate rapid proliferation 31fibroblasts during tissue repair and that the PDGF-RP, which is not afr-cted by these inflammatory mediators, is more Important to normal tissue maintainence.
IL-la PRODUCTION BY MONOCYTIC CELLS IS ENHANCED BY PLATELETS. Burton D. Clark, Ellen C. Donaldson, Koichi Aiura, Ronald G. Tompkins, Charles A. Dinarello. John F. Burke, and Jeffrey A. Gelfand. Dept. of.Meclicine, NEMC; Tufts Univ. School of Medicine: Mass. General Hospital; and Harvard Medical School, Boston, MA 02111 We have investigated the role of platelets with monocytes and their enhancement of cytokine production by the monocytic cell line, MonoMac 6. In cur studies, thrombin-activated platelets (P) were incubated with MonoMac 6 cells (M) at ratios of 0, 16, 80, 160, and 800 P/M with or without endotoxin (LPS) or heat-killed S. epidefmidis (SE). IL-la was quantitated by a specific and sensitive RIA subsequent to 4h and 8h incubations. The incubation of activated platelets with monocytic cells (16:l ratio) for 4h in the presence of LPS (M+P+LPS) at 50,100 and 500 pg/ml resulted in 3.6, 4.8, 4.9 fold increases in IL-la, respectively, compared to monocytes exposed to LPS without platelets (baseline, M+LPS). After 8h incubation, the overall percentage change from baseline decrease by approximately one-half, reflected by a corresponding decrease in baseline (M+LPS) cytokine production. Monocytes plus platelets incubated with S. epidermidis for 4 h (M+P+SE) showed a 3.5 fold increase over baseline (M+SE), and a 2 fold increase at 8h. There was a dose response for cytokine production with increased ratios of P:M. Overall, these results suggest that activated plateiets may magnify the cytokine response observed during both Gram-positive and Gram-negative infection.
A34
A31 INDUCIBLE EXPRESSION OF PTX3, A NEW MEMBER OF THE PENTRAXIN FAMILY, IN HUMAN MONONUCLEAR PHAGOCYTES. Barbara Bottazzi, VictorVidal Alles, Giuseppe Peri, JosCe Golay, Martin0 Introna and Albert0 Mantovani. Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy. PTX3 was recently cloned as an IL-l-inducible gene in endothelial cells, with structural similarities to pentraxins (C-reactive protein and Serum Amyloid P component). Aim of this study was to investigate the expression of PTX3 in human leukocytes populations. Human peripheral blood monocytes exposed to lipopolysaccaride or IL-lfi expressed significant levels of PTX3 messenger RNA (mRNA). TNFo. was a less effective inducer of PTX3, whereas IL-6, MCP-1, M-CSF, GM-CSF and IFN-7 were inactive. PTX3 transcript was undetectable in resting or stimulated polymorphonuclear cells, T or B lymphocytes and NK cells. PTX3 mRNA was also inducible in in vi&o monocytederived macrophages, tumor-associated macrophages and in the myelomonocytic cell lines HL60, U937 and THPl, but not in GFD8. Characterization of the binding properties of PTX3 is in progressusing a PTX3 specific antiserum, release of PTX3 protein was demonsrated for the first time in stimulated monocytes as well as in endothelial and fibroblastic cells. Thus, PTX3, unlike the classical pentraxins CRP and SAP, is expresed and released by cells of the monocyte-macrophage lineage exposed to inflammatory signals.
OF CHEMOKlNES, I.?, I.-H. Gongt, B. K. Rajaratbnam~, K.S. Kim%, and B.D. SykesP, TTbe Biomedical Research Centre and Biochemistry Department, UBC, Vancouver BC, V6T M; ‘Theodor-Kocher Institute, Uniwsity of Bern, Switzerland, CH 3CC0; ~Biochemishy Deparbnem, University of Alberta, Edmonton AB, T6G 2S2.
Recruitment of Mononuclear Cells to the Lung Using a Recombinant Adenovirus Expressing Murine RANTES. Todd A. Bra&k’, Zhou Xing’, Kevin Bacon’, Frank L. Graham’, Thomas J. Schall’, Carl D. Richards’, Ken Croitoru3 and Jack Gauldie’, Departments of Pathology’ and Medicine3, McMaster University, Hamilton, Canada L8N 325, DNAX Research Institute, Palo Alto, CA’. RANTES is a member of the C-C subfamily of chemokines that is reported to function as a chemoattractant for monocytes, eosinophils and CD4+ T cells. Using a recombinant human type 5 adenovirus (ADS) containing the murine RANTES cDNA (Ad5E3mRANTES), we initiated a study to characterize the biological functions of this cytokine in viva. Intratracheal administration with 2~10~ pfu of Ad5E3mRANTES or control virus targeted transgene expression to lung epithelial cells of Sprague-Dawley rats. Only Ad5E3mRANTES treated animals were found to have physiologic concentrations of RANTES cytokine detected in the bronchoalveolar lavage (BAL) fluids. Concurrent with the presence of RANTES cytokine were significant increases in the number of mononuclear cells. At 24h monocyte numbers were increased 50 fold in Ad5E3mRANTES treated rats. Histological examination of lung sections were consistent with the BAL data and indicates an active process of monocyte recruitment to the lung confirming the proinflammatory nature of RANTES in viva and its function as a monocyte chemoattractant. (Supported by MRC Canada)
QUANTIFICATION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST (IL-lra) GENE ALLELE-SPECIFIC TRANSCRIPTS IN KERATINOCYTES. F.E. Clay, J.K. Tarlow, MI. Cork and G.W. Duff. Section of Molecular Medicine, University of Sheffield, SlO 2JF, U.K. We have shown an allele of the IL-lra gene to be associated with the clinical outcome in several chronic inflammatory diseases. To test the functional correlate of this DNA variation, we measured mRNA from the two main alleles using a totally linked exonic marker. Allele 2 of this exonic polymorphism creates an Hpa II site, and is linked to the disease-related allele of IL-lra. RNA was isolated from keratinocytes and RT-PCR using a 5’ end-labelled primer was performed. The product was cut with Hpa II and analysed on a phosphorimager. The relative quantity of RNA from each allele was determined. In 9 of 12 heterozygotes, mRNA from allele 1 was significantly more abundant than from allele 2. Equal levels of mRNA were seen in 2 of 12 and 1 cell line showed more mRNA from allele 2 than from allele 1. This differential mRNA accumulation may be due to altered gene transcription or mRNA stability in the two alleles. Thus mRNA for this anti-inflammatory cytokine is less abundant in genotypes that have been associated with chronic inflammatory diseases.
S’IRUCTURE-FUNCTION Dewald*, M. Baggiolini*,
Functional and structural dialysis of interk&in-8 and other cbemokine analogs has provided insights into their recepmr interactions and stnxture-activity relatiombips. Interleukin-8. which has been shown to be a dim% was found to function as the monomeric form. Although struaml analysis revealed that MGSA is a dimcr in solution, it was found to dissociate to monomers at physiological concentrations. Analogs and CXC chemokine hybrids have shown &t &thin the IL-8 monomer, 4 smcbml motifs are immxtant: the N-terminal residues 4.5, and 6, termed the ELR motif; the N-tem&nJ ioop region that includes residues 1l-20; the 30-35 p turn; and tbe two disultide. bridges. It is hypothesized that the IL-8 protein, particularly the 30-35 p mm and the disultide bridges, provides a scaffold for orientation of the ELR motif, which is the primary receptor activation site. A Z-site receptor-binding model is proposed, similar to that suggested for CSa. In this model, receptor site 1 binds a secondary site of IL-8 comprising residues 11-20, whereas the primary receptor-binding site of IL-8, the ELR motif. binds a second, buried, receptor site. Results with MCP-1 analogs demons&ate the importance of the N-terminal region for hmction and, by analogy with IL-8, mmcated MCP-1 forms MCP-l-specific receptor antagonists. Thus, preliminary results with MCP-I suggest that the general principles established for the CXC chemckines will also apply to CC chemokines.
A32
A35 Tissue
Directed