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MCP-1: Cloning, expression, and protein secretion by monocytes
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POTENTIAL MECHANISM FOR THE RECRUITMENT OF LARGE GRANULAR LYMPHOCYTES (LGL) FOLLOWING TREATMENT WITH BIOLOGICAL RESPONSE MODIFIERS (BRM A.M. Pilarol.' and R.H. Wiltroutl lETS, LEI and 3.BCDP, PRI, NCI-FCRF, Frederick, MD 21701. Treatment of mice and rats with different BRM results in the To study the infiltration of activated IGL into the liver. mechanism by which I.GL are recruited, we have examined the BRM-induced release of chemotactic factors from cultures of hepatocytes (HC) and Kupffer cells (KC). Enriched rat peripheral blood LGL (>80%) were obtained by centrifugatio" on discontinuous Percoll gradients and chemotaxis was measured & vitro using a modified Boyden chamber assay. Treatment of isolated HC with either lipopolysaccharide (LPS,lO "g/ml) or acetsminophen (AA, 50$-Q resulted in the release of a supernatant factor(s) that stimulated LGL migration by 2-3 fold, while 10 “g/ml LPS or 100 pg/ml of the interferon inducer poly ICLC induced KC to produce a factor(s) that stimulated LGL chemotaxis by 30-70X. Similar results have been obtained using LGL and liver cells from mice. None of the BRM tested were chemotactic for rat or mouse LGL by themselves, suggesting that they act indirectly. To date we have found that several know" cytokines, including human recombinant (hr) interleukin-2 and purified ret interferon (IFN) u/p but not IFtQ or hr interleukin-8 induce a 3-5 fold increase in LGL migration at doses of 1000-3000 U/ml. These results suggest that the BRM-induced localization of LGL to the liver may partially be regulated by BRM-induced chemotactic factors.
BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HUMAN NATURAL GRANULOCYTE CHEMOTACTIC PROTEINIINTERLEUKIN-8 , J. Willem+ and M. Rampart+ J. Van Damme', B. Decock', 'Rega Institute and =Interdisciplinary Research Center, University of Leuven; +Laboratory of Experimental Pharmacology, University of Antwerp, Belgium A granulocyte chemotactic protein (GCP), recently designated interleukin-8 (IL-8) wes purified from stimulated human mononuclear cells. Upon heparin-sepharose chromatography GCP/IL-8 co-eluted with the structurally related platelet-derived B-thromboglobulin (BTG), but was separated from the latter by cation-exchange chromatography (FPLC). Both GCP/IL-8 and BTG proteins were present in multiple forms, differing in NH -terminal truncation. In addition to its chemotactic activi t y. GCP/IL-6 acted as a specific "eutrophil degranulator since it induced lactoferri" release from granules, and since it had less effect on enzyme rel.ease from primary granules. Furthermore, GCP/IL-8 exerted chemotactic activity in viva by inducing skin-reactivity and intragranulocytosis in rabbits. In the presence of PGE dermal injection of ng amounts of GCP/IL-8 resul %'ed in a fast (30 mi ) but short (t l/2 60-70 min) neutrophil accumu11P lation ( In-neutrophils) assoc&-ed with a paralleled plasma protein extravascation ( I-albumin). Similarly, intraveneous injection of GCP/IL-8 provoked a" immediate granulocytosis (60 min) whereas OTG did not show such effect and also lacked in vitro granulocyte chemotactic activity.
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THE HUMAN HOMOLOGUE OF IE IS MCAF/MCP-1: CLONING, EXPRESSION, AND PROkErN SECRETION BY MONOCYTES. Barrett I. Rollins. Peter Stier, Timothv Ernst. and Gordon G.Wong Dana-Farber Cancer Institute, Boston, MA 02115 and Genetics Institute, Inc., Cambridge, MA 02147. The ]E gene was one the first genes to be described whose expression is induced in mouse fibroblasts by PDGF. We have already shown that JE encodes a secreted glycoprotein (mJE) that belongs to a rapidly expanding family of low molecular weight chemotactic and inflammatory cytokines. To examine JE in human systems, we cloned the human homologue of JE from serum-stimulated WI38 cells. The protein predicted by the cDNA clone (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to the monocyte chemoattractant MCAF/MCP-1. When expressed in COS cells, the cDNA directs the secretion of proteins of Mr 13,000, 15,000, and 15,500, as well as N-glycosylated species of M, 16-l&$000. Antiserum raised against mJE recognizes these hJE species, all of which are secreted by human fibroblasts. Resting human monocytes constitutively secrete hJE; treatment with y-IFN does not enhance this secretion, and treatment with PMA or LPS inhibits hJE secretion. This fact plus the constitutive expression of mJE in mouse heart and cardiocytes suggest that JE protein may have other functions in addition to monocyte chemotaxis and activation.
THE GRANULOCYTE CHEMOTACTIC PROTEIN (GCP) FROM IL-l-INDUCED OR VIRALLY INFECTED FIBROBLASTS IS IDENTICAL TO MONOCYTEDERIVED GCP/IL-8 J. Van Damme, R. Conings, B. Decock, J.-P. Lenaerts, G. Opdenakker and A. Billiau Rega InsSitute, University of Leuven, Belgium IL-l does not exert chemotactic activity in vitro and induces skin-reactivity and granulocytosis in rabbits with slower kinetics than GCPIIL-8. This suggests that the latter could be a mediator for these in viva effects of IL-I. This hypothesis was supported by the finding that normal skin fibroblasts stimulated with IL-l@ produced a chemotactic factor for granulocytes. This activity was purified to bomogeneity by successive adsorption to controlled pore glass, hepsrin-sepharose chromatography and FPLC. The factor showed en NH*-terminal sequence identical to that of GCPIIL-8 from monocytes. Moreover, a specific artibody against purified GCP/IL-8 from monocytes could immunoprecipitate and "eutralize pure GCP from fibroblasts. Furthermore, GCP/IL-8 was also induced in normal fibroblasts and on a" osteosarcoma cell line by the double-stranded RNA poly rI:rC, as well as by pathogens such as measles and rubella virus. It can thus be concluded that GCPIIL-8 is produced by several cell types in response to IL-l or infectious agents and that fibroblasts can contribute to chemotaxis in inflammation.
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RECOHBINANT HUMAN NAP-l/IL-B SHOWS DIFFERING CHEHOTACTIC AND STIHULATORY ACTIVITY ON CELLS FROM DIFFERENT SPECIES. Antal Rot, Charles Lam and Ivan J.D. Lindley. Sandoz Forschungsinstitut, A-1235 Vienna, Austria
CLDNINGOF'ITiEI%XJSEHCPIOLM;OF IP-10 ~LYMFMOKINEACPIVATEU R&W 264.7 CZXS. P. Vansuri and J. Farber. Johns Hopkins Univ. sch. of Med., Balttira, MD 21205 Ac~lib?z&ywasrpadefnwthe mouse lracrophage-like cell line RAW 264.7 stinn&ited for 3 hours with medium from c~~a~valin A-treated spleen cells, and screened usingprobe prepared frcsnatiimllateflandcXXltro1ce11s. cIX??+clo"eawere isolated that coded for a 98 amino acid protein that was identical to the IFN-Yin&c&h-protein IPlO at 68 positions. l% m for the mouse protein was approximately 1.4 Kb in length by Northern analysis and was detectable at 30 minutes after stirrmlation with IF!+ y , reaching nz&nal levels between 2 ard 6 hours. The accumulation of n@NA did not require new protein synthesis. Besides IFN-Y, IFN-[cr & 81 and er&toxin were also able to induce accumulation of the mRNA. The n@NA was inducible in starch-elicited peritoneal nmxophages as well as in RAW cells. In addition to the coding sequence homolcgy there was extensive nucleotide conservation in portions of the 3'-untra"slated regions of themouseardhuman~ences. Themuse cIX?Accdes for a protein of 10,781 m1t.o" whose amino terminus includes, as is the case for IP-10, a predicted signal peptide of 21 amino acids. 'Iheenccdedproteinispresumed to be the murine homolcg of IPlO, a qtokine of unknc%n function that is a recently discovered member of the platelet factor 4 family of secretedproteins. supportedinpartbyqrantnumberR29 CA48059 frcan the NCI.
The species-specificity of many human cytokines can be a major barrier to complete understanding of their role in viva and their position in the cytokine network, since many animal models are rendered unusable by lack of activity. Study of cross-species reactivity, therefore, can yield important insights into the range of models available. We have examined the ability of our recombinant human NAP-l/IL-8 to induce chemotaxis and respiratory burst in the PMNs of 8 species: human, rat, mouse, pig, dog, rabbit, guinea pig and monkey. PMNs from all species tested responded in the chemotaxis assay, albeit with differing magnitudes and optimal concentrations of ranging from recombinant material, 100 rig/ml to 20 ug/ml. Similar species differences were observed in the respiratory burst assay, and we have correlated these in vitro observations in some cases with in viva results.
INTERNATIONAL
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ON CYTOKINES
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POTENTIAL MECHANISM FOR THE RECRUITMENT OF LARGE GRANULAR LYMPHOCYTES (LGL) FOLLOWING TREATMENT WITH BIOLOGICAL RESPONSE MODIFIERS (BRM A.M. Pilarol.' and R.H. Wiltroutl lETS, LEI and 3.BCDP, PRI, NCI-FCRF, Frederick, MD 21701. Treatment of mice and rats with different BRM results in the To study the infiltration of activated IGL into the liver. mechanism by which I.GL are recruited, we have examined the BRM-induced release of chemotactic factors from cultures of hepatocytes (HC) and Kupffer cells (KC). Enriched rat peripheral blood LGL (>80%) were obtained by centrifugatio" on discontinuous Percoll gradients and chemotaxis was measured & vitro using a modified Boyden chamber assay. Treatment of isolated HC with either lipopolysaccharide (LPS,lO "g/ml) or acetsminophen (AA, 50$-Q resulted in the release of a supernatant factor(s) that stimulated LGL migration by 2-3 fold, while 10 “g/ml LPS or 100 pg/ml of the interferon inducer poly ICLC induced KC to produce a factor(s) that stimulated LGL chemotaxis by 30-70X. Similar results have been obtained using LGL and liver cells from mice. None of the BRM tested were chemotactic for rat or mouse LGL by themselves, suggesting that they act indirectly. To date we have found that several know" cytokines, including human recombinant (hr) interleukin-2 and purified ret interferon (IFN) u/p but not IFtQ or hr interleukin-8 induce a 3-5 fold increase in LGL migration at doses of 1000-3000 U/ml. These results suggest that the BRM-induced localization of LGL to the liver may partially be regulated by BRM-induced chemotactic factors.
BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HUMAN NATURAL GRANULOCYTE CHEMOTACTIC PROTEINIINTERLEUKIN-8 , J. Willem+ and M. Rampart+ J. Van Damme', B. Decock', 'Rega Institute and =Interdisciplinary Research Center, University of Leuven; +Laboratory of Experimental Pharmacology, University of Antwerp, Belgium A granulocyte chemotactic protein (GCP), recently designated interleukin-8 (IL-8) wes purified from stimulated human mononuclear cells. Upon heparin-sepharose chromatography GCP/IL-8 co-eluted with the structurally related platelet-derived B-thromboglobulin (BTG), but was separated from the latter by cation-exchange chromatography (FPLC). Both GCP/IL-8 and BTG proteins were present in multiple forms, differing in NH -terminal truncation. In addition to its chemotactic activi t y. GCP/IL-6 acted as a specific "eutrophil degranulator since it induced lactoferri" release from granules, and since it had less effect on enzyme rel.ease from primary granules. Furthermore, GCP/IL-8 exerted chemotactic activity in viva by inducing skin-reactivity and intragranulocytosis in rabbits. In the presence of PGE dermal injection of ng amounts of GCP/IL-8 resul %'ed in a fast (30 mi ) but short (t l/2 60-70 min) neutrophil accumu11P lation ( In-neutrophils) assoc&-ed with a paralleled plasma protein extravascation ( I-albumin). Similarly, intraveneous injection of GCP/IL-8 provoked a" immediate granulocytosis (60 min) whereas OTG did not show such effect and also lacked in vitro granulocyte chemotactic activity.
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449
THE HUMAN HOMOLOGUE OF IE IS MCAF/MCP-1: CLONING, EXPRESSION, AND PROkErN SECRETION BY MONOCYTES. Barrett I. Rollins. Peter Stier, Timothv Ernst. and Gordon G.Wong Dana-Farber Cancer Institute, Boston, MA 02115 and Genetics Institute, Inc., Cambridge, MA 02147. The ]E gene was one the first genes to be described whose expression is induced in mouse fibroblasts by PDGF. We have already shown that JE encodes a secreted glycoprotein (mJE) that belongs to a rapidly expanding family of low molecular weight chemotactic and inflammatory cytokines. To examine JE in human systems, we cloned the human homologue of JE from serum-stimulated WI38 cells. The protein predicted by the cDNA clone (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to the monocyte chemoattractant MCAF/MCP-1. When expressed in COS cells, the cDNA directs the secretion of proteins of Mr 13,000, 15,000, and 15,500, as well as N-glycosylated species of M, 16-l&$000. Antiserum raised against mJE recognizes these hJE species, all of which are secreted by human fibroblasts. Resting human monocytes constitutively secrete hJE; treatment with y-IFN does not enhance this secretion, and treatment with PMA or LPS inhibits hJE secretion. This fact plus the constitutive expression of mJE in mouse heart and cardiocytes suggest that JE protein may have other functions in addition to monocyte chemotaxis and activation.
THE GRANULOCYTE CHEMOTACTIC PROTEIN (GCP) FROM IL-l-INDUCED OR VIRALLY INFECTED FIBROBLASTS IS IDENTICAL TO MONOCYTEDERIVED GCP/IL-8 J. Van Damme, R. Conings, B. Decock, J.-P. Lenaerts, G. Opdenakker and A. Billiau Rega InsSitute, University of Leuven, Belgium IL-l does not exert chemotactic activity in vitro and induces skin-reactivity and granulocytosis in rabbits with slower kinetics than GCPIIL-8. This suggests that the latter could be a mediator for these in viva effects of IL-I. This hypothesis was supported by the finding that normal skin fibroblasts stimulated with IL-l@ produced a chemotactic factor for granulocytes. This activity was purified to bomogeneity by successive adsorption to controlled pore glass, hepsrin-sepharose chromatography and FPLC. The factor showed en NH*-terminal sequence identical to that of GCPIIL-8 from monocytes. Moreover, a specific artibody against purified GCP/IL-8 from monocytes could immunoprecipitate and "eutralize pure GCP from fibroblasts. Furthermore, GCP/IL-8 was also induced in normal fibroblasts and on a" osteosarcoma cell line by the double-stranded RNA poly rI:rC, as well as by pathogens such as measles and rubella virus. It can thus be concluded that GCPIIL-8 is produced by several cell types in response to IL-l or infectious agents and that fibroblasts can contribute to chemotaxis in inflammation.
441
450
RECOHBINANT HUMAN NAP-l/IL-B SHOWS DIFFERING CHEHOTACTIC AND STIHULATORY ACTIVITY ON CELLS FROM DIFFERENT SPECIES. Antal Rot, Charles Lam and Ivan J.D. Lindley. Sandoz Forschungsinstitut, A-1235 Vienna, Austria
CLDNINGOF'ITiEI%XJSEHCPIOLM;OF IP-10 ~LYMFMOKINEACPIVATEU R&W 264.7 CZXS. P. Vansuri and J. Farber. Johns Hopkins Univ. sch. of Med., Balttira, MD 21205 Ac~lib?z&ywasrpadefnwthe mouse lracrophage-like cell line RAW 264.7 stinn&ited for 3 hours with medium from c~~a~valin A-treated spleen cells, and screened usingprobe prepared frcsnatiimllateflandcXXltro1ce11s. cIX??+clo"eawere isolated that coded for a 98 amino acid protein that was identical to the IFN-Yin&c&h-protein IPlO at 68 positions. l% m for the mouse protein was approximately 1.4 Kb in length by Northern analysis and was detectable at 30 minutes after stirrmlation with IF!+ y , reaching nz&nal levels between 2 ard 6 hours. The accumulation of n@NA did not require new protein synthesis. Besides IFN-Y, IFN-[cr & 81 and er&toxin were also able to induce accumulation of the mRNA. The n@NA was inducible in starch-elicited peritoneal nmxophages as well as in RAW cells. In addition to the coding sequence homolcgy there was extensive nucleotide conservation in portions of the 3'-untra"slated regions of themouseardhuman~ences. Themuse cIX?Accdes for a protein of 10,781 m1t.o" whose amino terminus includes, as is the case for IP-10, a predicted signal peptide of 21 amino acids. 'Iheenccdedproteinispresumed to be the murine homolcg of IPlO, a qtokine of unknc%n function that is a recently discovered member of the platelet factor 4 family of secretedproteins. supportedinpartbyqrantnumberR29 CA48059 frcan the NCI.
The species-specificity of many human cytokines can be a major barrier to complete understanding of their role in viva and their position in the cytokine network, since many animal models are rendered unusable by lack of activity. Study of cross-species reactivity, therefore, can yield important insights into the range of models available. We have examined the ability of our recombinant human NAP-l/IL-8 to induce chemotaxis and respiratory burst in the PMNs of 8 species: human, rat, mouse, pig, dog, rabbit, guinea pig and monkey. PMNs from all species tested responded in the chemotaxis assay, albeit with differing magnitudes and optimal concentrations of ranging from recombinant material, 100 rig/ml to 20 ug/ml. Similar species differences were observed in the respiratory burst assay, and we have correlated these in vitro observations in some cases with in viva results.