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Induction of IL-6 expression in and by human mast cells following cellular activation
546
I Abstracts
CYTOKINE,
P3 October 2: In.ammation
Vol. 6, No. 5 (September
1994: 539-582)
and Cytokines
A42
A45 BIDIRECTIONAL INTERACTIONS OF GP130 CYTOKINES AND THE NEUROENDOCRINE SYSTEM P. Ghezzi, F. Benigni, G. Fantuzzi Istituto “Mario Negri”, Via Eritrea 62, 20157 Milan,Italy Activation of the hypothalamus-pituitary-adrenal axis (HP& is a major feedback mechanism controlling the production and action of cytokines, and injection of LPS,TNF or IL-l increases serum corticosterone (CS) levels. We observed higher LPS toxicity and TNF production in mice where the HPAA is blocked by adrenaiectomy 01‘ desensitized by glucocorticoid (GC) pretreatment (a rebound effect associated with GC therapy). On the contrary, whole blood obtained from stressed mice shows an inhibited TNF production, which is restored by GC receptor antagonists, indicating that endogenous GC can actually inhibit TNF production. Cytokinesjneurotrophic factors acting through gp130 (CNTF, IL-II, IL-6, Oncostatin M), while not increasing CS per se, potentiate the elevation of serum CS by a concomitant injection of a suboptimal dose of IL-l. This activation is often associated with inhibited TNF production and limits LPS toxicity.
CHARACTERIZATION OF IL-IBETA, IL-6 AND TNF IN THE VAGINA DURING PREGNANCY P. Greig, H.lmseis and C. Livengood, J. Duke Univ. Med. Cntr., Durham, NC 27710 Cytokines produced in response to infection have been implicated in the etiology of preterm birth. Bacterial vaginosis (BV) has also been associated with preterm birth. Since the presence of inflammatory cytoklnes in the vagina has not been well described, we sought to characterize the presence of interleukin-lbeta (IL-ibeta), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in the vagina during pregnancy in patients with and without BV. Vaginal fluid was obtained from pregnant women (N=45) by lavage. Cytokine levels were measured by ELISA and are expressed in pglml. There was a very wide range of vaginal cytokine levels detected in the study patients (IL-lbeta: Range = O-512, Mean = 247, Median = 216; IL-6: Range = O-681,703, Mean = 37,961, Median = 0; TNF: Range = O-90,401, Mean = 9,904, Median = 6,938). Vaginal levels of IL-1 beta were significantly higher (pc.05) in the BV group (N=i4) compared to vaginal levels from patients without BV (N=31). There were no significant differences between the vaginal IL-6 and TNF levels found in these groups. Conclusions: (1) Significant quantities of inflammatory cytokines were found in the vagina during pregnancy. (2) There was very poor correlation between the three different cytokine levels from each patient. (3) Only IL-lbeta levels were found to be significantly elevated in the vaginal wash samples of patients with BV. These data pose some interesting questions as to the role and source of cytokines in the vagina during pregnancy.
two
A43
A46 Antagonists of Monocyte Chemoattractant Protein(MCP-1) Identified by Modification of Functionally CriUcal NHz-Terminal Residues Jiang-Hong Gong and Ian Clark-Lewis ‘Ihe Biomedical Research Centre. UBC. Vancouver
1
BC, V6T 123
Distinct patterns of organ expression normal homeostasis or inflammatory
of IL-1 versus IL-6 and &F-l states.
in
Moshe Hacham. Natalio Crystal, Shraga Segal and Ron N. Apte, Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University, Bee&hew 84105, Israel.
MCP-1 analogs were designed to determine the role of the NH2terminal region in structure and function. The NH2-terminal residue at position 1 could not be deleted or extended. However the NH2-terminal pyroglutamate of the wild type was not essential as it could be replaced by several other non-cycIic amino acids without loss of activity. Residues 7-10 were essential for receptor desensitization. but were not sufficient for function. and -the integritv of residures 1-6 were reauired for biological acttvlty. Nei&er-MCP-l(1 l-76) nor the MCP-1‘(1-10) peptide &d detectable ago&& or MCP-1 antagonist acuvity , indicating that residues l-10 are essential. but not sufficient. for function and that other MCP-1 residues are also involved. Several truncated analogs, MCP-1 7-76. 8-76. 9-76 and lo-76 desensitized MCP-1 induced monocytes Ca2+ efvation and chemotaxis. but did not have activity by them own. These analogs antagonIzed MCP-1 induced response with the most potent being MCP-1 9-76 [IC50=6O III@. The 9-76 analog specifically inhibited the calcium mobilization induced bv MCP-1, MCP-2 and MCP-3. but not that of other CC chemokinks. suggesting that it is MCP receptor specific. The availability of these compounds will be helpful in evaluating MCP-1 receptor antagonists as anti- Ulammatoxy therepeutics.
A distinct pattern of IL-l, IL-6 and CSF-1 expression is depicted in organs from conventional or LPS-treated mice; the latter repiesenting an inflammatory stimulus. Specific cytokine bioassays of organderived conditioned media or lysates were applied. A mirror-image relationshio emewes between fhe o&terns of IL-I versus IL-6 and CSF1 express& in t’wo groups of &gans: those with lymphoreticular function (liver, spleen, intestine and lungs), which manifest high IL-1 and low IL-6/C%1 activity, as compared to organs characterized by highly specialized and potentially vulnerable functions (such as the heart, brain, muscle and kidney), which exhibit high ILd/CSF-1 and low IL-l activity. Thus, lymphoreticular organs, due to their defensive functions, mount IL-l-mediated extensive inflammatory responses, even with the cost of tissue damage. In contrast, indispensable internal organs mount ILd/CSF-l-mediated responses which are milder and bear less potential of tissue damage. Characterization of distinct patterns of expression of proinflammatory cytokines in differ&t organs, at steady state or udder inflammatory conditions, may shed light on tissue unique homeostatic and defensive mechanisms.
INLNJCTIONOF IL-6 EXPRESSIOWIN AND BY HUlwl MST CELLS FOLLORINS CELLULARACTIVATION. John R. 6wdon5. Cheryl Rousel, Anne-Marie A. Ireni(. and Lawrence 6. Schnartz~. slept. Vet. Microbial., University of Saskatchewan, Saskatoon, Canada, and nthe Dept. Iled.. Rdical College of Virginia, Virginia Cannn*ealth University, Richnmnd. VA. Me used in situ hybridization (ISH) to examine the expression of IL-6 mRNAin in vitro-derived human mast cells stimulated with phorbol esters and in human skin biopsies sensitized with IgE and challenged with a nonoclanal anti-IgE antibody (HR121) ex viva Humanfetal liver cells grown for 4 weeks in SO ngA1 reccvnbinant human stem cell factor (SCF) comprise 2 602 mast cells as determined by toluidine blue and tryptase staining. Following stiwlation with phorbol myristate acetate (FM; SO nghl). the mast cells in these populations expressed very high levels of IL-6 mRRA. whereas time-matched control mest cells not stimulated with pborbol esters expressed only low levels of IL-6 SIRNA. In a second series of experiments. human skin samples from 3-4 day neonates were sensitized with Igf antibody, washed and either challenged with nmnoclonal antibody HBlZl or left unstimulated (negative cantml). At varying times following the addition of antiIgE, the culture supematants *em assayed for TNF content and the tissues were recessed for 1% Ye observed a significant release of lNFd(p
INSULIN ALTERATION OFTNF AND IL-6 PRODUCTION INVOLVES PHOSPHATASE REGULATION. E.L. Hahn, E.J. Kovacs and J.P. Filkins. Loyola University of Chicago, Maywood, IL 60153. We have recently demonstrated that insulin (1) alters endotoxin
A44
These results
indicate
that in vitm-derived
human mast cells
express IL-6 mRNA at very high levels following stimulation with PM. They also suggest that activation of cutaneous hunan mast cells via the FqRI leads to high level IL-6 production in peripheral tissues.
stimulation and especially dexamethasone (D) suppression of macrophage TNF and IL-6 production. To elucidate if l’s influence occurs via phosphatase regulation, peritoneal and ANA1 macrophages were pretreated with (D), 3 hours prior to the addition of S. enteritidis endotoxin (E). One hour after E, I and okedaic acid (0) - an inhibitor of phosphatases 1 and 2A, were added to the cells. The media was harvested 18 hours after insulin addition and analyzed for TNF and IL-6. 0 treatment stimulated TNF and IL-6 production by ANA1 and peritoneal macrophages. 0 enhanced E-stimulated TNF and IL-6 production by both cell types. E+I suppressed both TNF and IL-6 production by ANA1 cells but did not alter peritoneal macrophage TNF or IL-6 production. E + I + 0 resulted in significantly enhanced TNF and IL-6 production by ANA1 cells. D suppression of IL-6 production by ANA1 cells and TNF and IL-6 production by peritoneal macrophages was reversed by 0 addition. D+E+I treatment resulted in reestablished TNF and IL-6 production 0 treatment of this group suppressed only TNF production by peritoneal macrophages and had no effect on IL-6 oroduction bv ANA1 or oeritoneal macrophages. Therefore, I alteration of TiF and IL-6 probuction in macrophages appears to involve the differential regulation of phosphatase activity.
I Abstracts
CYTOKINE,
P3 October 2: In.ammation
Vol. 6, No. 5 (September
1994: 539-582)
and Cytokines
A42
A45 BIDIRECTIONAL INTERACTIONS OF GP130 CYTOKINES AND THE NEUROENDOCRINE SYSTEM P. Ghezzi, F. Benigni, G. Fantuzzi Istituto “Mario Negri”, Via Eritrea 62, 20157 Milan,Italy Activation of the hypothalamus-pituitary-adrenal axis (HP& is a major feedback mechanism controlling the production and action of cytokines, and injection of LPS,TNF or IL-l increases serum corticosterone (CS) levels. We observed higher LPS toxicity and TNF production in mice where the HPAA is blocked by adrenaiectomy 01‘ desensitized by glucocorticoid (GC) pretreatment (a rebound effect associated with GC therapy). On the contrary, whole blood obtained from stressed mice shows an inhibited TNF production, which is restored by GC receptor antagonists, indicating that endogenous GC can actually inhibit TNF production. Cytokinesjneurotrophic factors acting through gp130 (CNTF, IL-II, IL-6, Oncostatin M), while not increasing CS per se, potentiate the elevation of serum CS by a concomitant injection of a suboptimal dose of IL-l. This activation is often associated with inhibited TNF production and limits LPS toxicity.
CHARACTERIZATION OF IL-IBETA, IL-6 AND TNF IN THE VAGINA DURING PREGNANCY P. Greig, H.lmseis and C. Livengood, J. Duke Univ. Med. Cntr., Durham, NC 27710 Cytokines produced in response to infection have been implicated in the etiology of preterm birth. Bacterial vaginosis (BV) has also been associated with preterm birth. Since the presence of inflammatory cytoklnes in the vagina has not been well described, we sought to characterize the presence of interleukin-lbeta (IL-ibeta), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in the vagina during pregnancy in patients with and without BV. Vaginal fluid was obtained from pregnant women (N=45) by lavage. Cytokine levels were measured by ELISA and are expressed in pglml. There was a very wide range of vaginal cytokine levels detected in the study patients (IL-lbeta: Range = O-512, Mean = 247, Median = 216; IL-6: Range = O-681,703, Mean = 37,961, Median = 0; TNF: Range = O-90,401, Mean = 9,904, Median = 6,938). Vaginal levels of IL-1 beta were significantly higher (pc.05) in the BV group (N=i4) compared to vaginal levels from patients without BV (N=31). There were no significant differences between the vaginal IL-6 and TNF levels found in these groups. Conclusions: (1) Significant quantities of inflammatory cytokines were found in the vagina during pregnancy. (2) There was very poor correlation between the three different cytokine levels from each patient. (3) Only IL-lbeta levels were found to be significantly elevated in the vaginal wash samples of patients with BV. These data pose some interesting questions as to the role and source of cytokines in the vagina during pregnancy.
two
A43
A46 Antagonists of Monocyte Chemoattractant Protein(MCP-1) Identified by Modification of Functionally CriUcal NHz-Terminal Residues Jiang-Hong Gong and Ian Clark-Lewis ‘Ihe Biomedical Research Centre. UBC. Vancouver
1
BC, V6T 123
Distinct patterns of organ expression normal homeostasis or inflammatory
of IL-1 versus IL-6 and &F-l states.
in
Moshe Hacham. Natalio Crystal, Shraga Segal and Ron N. Apte, Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University, Bee&hew 84105, Israel.
MCP-1 analogs were designed to determine the role of the NH2terminal region in structure and function. The NH2-terminal residue at position 1 could not be deleted or extended. However the NH2-terminal pyroglutamate of the wild type was not essential as it could be replaced by several other non-cycIic amino acids without loss of activity. Residues 7-10 were essential for receptor desensitization. but were not sufficient for function. and -the integritv of residures 1-6 were reauired for biological acttvlty. Nei&er-MCP-l(1 l-76) nor the MCP-1‘(1-10) peptide &d detectable ago&& or MCP-1 antagonist acuvity , indicating that residues l-10 are essential. but not sufficient. for function and that other MCP-1 residues are also involved. Several truncated analogs, MCP-1 7-76. 8-76. 9-76 and lo-76 desensitized MCP-1 induced monocytes Ca2+ efvation and chemotaxis. but did not have activity by them own. These analogs antagonIzed MCP-1 induced response with the most potent being MCP-1 9-76 [IC50=6O III@. The 9-76 analog specifically inhibited the calcium mobilization induced bv MCP-1, MCP-2 and MCP-3. but not that of other CC chemokinks. suggesting that it is MCP receptor specific. The availability of these compounds will be helpful in evaluating MCP-1 receptor antagonists as anti- Ulammatoxy therepeutics.
A distinct pattern of IL-l, IL-6 and CSF-1 expression is depicted in organs from conventional or LPS-treated mice; the latter repiesenting an inflammatory stimulus. Specific cytokine bioassays of organderived conditioned media or lysates were applied. A mirror-image relationshio emewes between fhe o&terns of IL-I versus IL-6 and CSF1 express& in t’wo groups of &gans: those with lymphoreticular function (liver, spleen, intestine and lungs), which manifest high IL-1 and low IL-6/C%1 activity, as compared to organs characterized by highly specialized and potentially vulnerable functions (such as the heart, brain, muscle and kidney), which exhibit high ILd/CSF-1 and low IL-l activity. Thus, lymphoreticular organs, due to their defensive functions, mount IL-l-mediated extensive inflammatory responses, even with the cost of tissue damage. In contrast, indispensable internal organs mount ILd/CSF-l-mediated responses which are milder and bear less potential of tissue damage. Characterization of distinct patterns of expression of proinflammatory cytokines in differ&t organs, at steady state or udder inflammatory conditions, may shed light on tissue unique homeostatic and defensive mechanisms.
INLNJCTIONOF IL-6 EXPRESSIOWIN AND BY HUlwl MST CELLS FOLLORINS CELLULARACTIVATION. John R. 6wdon5. Cheryl Rousel, Anne-Marie A. Ireni(. and Lawrence 6. Schnartz~. slept. Vet. Microbial., University of Saskatchewan, Saskatoon, Canada, and nthe Dept. Iled.. Rdical College of Virginia, Virginia Cannn*ealth University, Richnmnd. VA. Me used in situ hybridization (ISH) to examine the expression of IL-6 mRNAin in vitro-derived human mast cells stimulated with phorbol esters and in human skin biopsies sensitized with IgE and challenged with a nonoclanal anti-IgE antibody (HR121) ex viva Humanfetal liver cells grown for 4 weeks in SO ngA1 reccvnbinant human stem cell factor (SCF) comprise 2 602 mast cells as determined by toluidine blue and tryptase staining. Following stiwlation with phorbol myristate acetate (FM; SO nghl). the mast cells in these populations expressed very high levels of IL-6 mRRA. whereas time-matched control mest cells not stimulated with pborbol esters expressed only low levels of IL-6 SIRNA. In a second series of experiments. human skin samples from 3-4 day neonates were sensitized with Igf antibody, washed and either challenged with nmnoclonal antibody HBlZl or left unstimulated (negative cantml). At varying times following the addition of antiIgE, the culture supematants *em assayed for TNF content and the tissues were recessed for 1% Ye observed a significant release of lNFd(p
INSULIN ALTERATION OFTNF AND IL-6 PRODUCTION INVOLVES PHOSPHATASE REGULATION. E.L. Hahn, E.J. Kovacs and J.P. Filkins. Loyola University of Chicago, Maywood, IL 60153. We have recently demonstrated that insulin (1) alters endotoxin
A44
These results
indicate
that in vitm-derived
human mast cells
express IL-6 mRNA at very high levels following stimulation with PM. They also suggest that activation of cutaneous hunan mast cells via the FqRI leads to high level IL-6 production in peripheral tissues.
stimulation and especially dexamethasone (D) suppression of macrophage TNF and IL-6 production. To elucidate if l’s influence occurs via phosphatase regulation, peritoneal and ANA1 macrophages were pretreated with (D), 3 hours prior to the addition of S. enteritidis endotoxin (E). One hour after E, I and okedaic acid (0) - an inhibitor of phosphatases 1 and 2A, were added to the cells. The media was harvested 18 hours after insulin addition and analyzed for TNF and IL-6. 0 treatment stimulated TNF and IL-6 production by ANA1 and peritoneal macrophages. 0 enhanced E-stimulated TNF and IL-6 production by both cell types. E+I suppressed both TNF and IL-6 production by ANA1 cells but did not alter peritoneal macrophage TNF or IL-6 production. E + I + 0 resulted in significantly enhanced TNF and IL-6 production by ANA1 cells. D suppression of IL-6 production by ANA1 cells and TNF and IL-6 production by peritoneal macrophages was reversed by 0 addition. D+E+I treatment resulted in reestablished TNF and IL-6 production 0 treatment of this group suppressed only TNF production by peritoneal macrophages and had no effect on IL-6 oroduction bv ANA1 or oeritoneal macrophages. Therefore, I alteration of TiF and IL-6 probuction in macrophages appears to involve the differential regulation of phosphatase activity.