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S Stafford

Publications and source records attributed to S Stafford.

At least 19 recordsLinked to original sources
PubMed2005

An NCIC-CTG phase I dose escalation pharmacokinetic study of the matrix metalloproteinase inhibitor BAY 12-9566 in combination with doxorubicin.

BACKGROUND: This phase I study was performed to evaluate the safety, tolerability, and efficacy of the oral matrix metalloproteinase inhibitor BAY 12-9566 in combination with doxorubicin in patients with advanced solid tumours, and to identify the maximum tolerated dose of these agents in combination and the dose for use in subsequent studies. PATIENTS AND METHODS: 14 patients were entered onto 3 dose levels consisting of escalating doses of doxorubicin (50 mg/m(2), 60 mg/m(2) and 70 mg/m(2)) with 800 mg po bid BAY 12-9566. At all three dose levels, patients received doxorubicin alone in cycle one on day 1. Daily oral dosing with BAY 12-9566 was started on day 8 of cycle 1, and thus doxorubicin was given concurrently with BAY 12-9566 in cycle 2. Patients were continued on treatment until a dose limiting toxicity or tumour progression occurred. RESULTS: Pharmacokinetic studies from cycles 1 and 2 from the patients treated in the first three dose levels demonstrated that the addition of BAY 12-9566 increased the AUC(0-12h) levels of doxorubicin by a median of 48%. No effects were seen on the BAY 12-9566 pharmacokinetic values. Two dose limiting toxicities were seen at the third dose level. One patient experienced grade 3 stomatitis in cycle 2, and another patient experienced grade 4 granulocytopenia in cycle 1 and grade 4 thrombocytopenia in cycle 2. Thus the maximum tolerated dose of 60 mg/m(2) was declared. These toxicities were those that would have been expected from doxorubicin alone. CONCLUSIONS: BAY 12-9566 can be safely administered with full doses of doxorubicin without evidence of clinical interaction. The recommended dose of doxorubicin to be combined with BAY 12-9566 800 mg po b.i.d is 60 mg/m(2), however, further development of BAY 12-9566 has been abandoned.

Adult↗
PubMed2005

A phase I study of oral ZD 1839 given daily in patients with solid tumors: IND.122, a study of the Investigational New Drug Program of the National Cancer Institute of Canada Clinical Trials Group.

PURPOSE: To define the maximum tolerated dose (MTD), the dose limiting toxicity (DLT), the biological active (BA) dose and the pharmacokinetics (PK) of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa) when administered continuously as a once daily dose in patients with advanced, incurable solid tumours. PATIENTS AND METHODS: Twenty-eight patients were enrolled in cohorts of three from three National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) centers. ZD1839 was given at doses from 150 to 800 mg daily orally and patients underwent a pretreatment and a 28 day post treatment tumor biopsy, while PK sampling was performed on days 8, 15, 22, 29, and a toxicity assessment every 28 days. RESULTS: All twenty-eight patients were evaluable for non-hematological and hematological toxicity. Twenty-seven were evaluable for response. The MTD was not reached but DLT included reversible rash and diarrhea. One patient with urachal cancer had a transient 55% decrease in tumor size and two other patients (breast and non-small cell lung cancer) had minor responses; three additional patients had pharmacodynamic evidence of target effect. PK demonstrated steady state within the first 2 weeks of dosing and dose dependent exposure. CONCLUSION: It appears that ZD 1839 at a dose of 800 m/day was tolerable, although some patients required dose modification for diarrhea. Doses above 250 m/day demonstrate biologic activity and could be consider for future study in a variety of EGFR positive tumor types.

Administration, Oral↗
PubMed2005

An NCIC CTG phase I/pharmacokinetic study of the matrix metalloproteinase and angiogenesis inhibitor BAY 12-9566 in combination with 5-fluorouracil/leucovorin.

BACKGROUND: This phase I study was performed to evaluate the safety, tolerability, and efficacy of the oral matrix metalloproteinase inhibitor BAY 12-9566 in combination with 5-fluorouracil/leucovorin in patients with advanced solid tumours, and to identify the maximum tolerated dose and the dose for use in future studies. PATIENTS AND METHODS: BAY 12-9566 and 5-fluorouracil/leucovorin were administered to 17 patients in 3 cohorts. Each patient served as his/her own control, with 5-fluorouracil being given alone on days 1-5 of cycle 1. In cohort 1, BAY 12-9566 at 800 mg p.o. b.i.d. was given with 350 mg/m2 5-fluorouracil/20 mg/m2 leucovorin x 5 days q28 days. In cohort 2, the BAY 12-9566 dose was reduced to 400 mg p.o. b.i.d., with the 5-fluorouracil/leucovorin doses remaining unchanged. Finally, in cohort 3, BAY 12-9566 400 mg bid was given with 5-fluorouracil 400 mg/m2/day. Patients were continued on therapy until unacceptable toxicity or tumour progression occurred. Pharmacokinetic analyses for both BAY 12-9566 and 5-fluorouracil were performed. RESULTS: The maximum tolerated dose was 400 mg p.o. b.i.d. BAY 12-9566 plus 5-fluorouracil/leucovorin at 400 mg/m2/day and 20 mg/m2/day, respectively. Thrombocytopenia necessitated a decrease of the dose of BAY 12-9566 by 50% from cohort 1 to cohort 2. Two dose-limiting toxicities occurred in cohort 3 consisting of neutropenic fever, and ileitis, causing severe diarrhea. Of 17 patients treated on study, 7 of 14 patients evaluable for response achieved stable disease. Pharmacokinetic analysis suggested there was no interaction between BAY 12-9566 and 5-fluorouracil. CONCLUSIONS: BAY 12-9566 400 mg bid and 5-fluorouracil 350 mg/m2 plus leucovorin 20 mg/m2 can be co-administered. Although there is some evidence of a clinical interaction, there is no apparent pharmacokinetic interaction. Future studies with these 2 types of agents administered in combination are warranted.

Angiogenesis Inhibitors↗
PubMed2002

Interleukin-12 inhibits eosinophil differentiation from bone marrow stem cells in an interferon-gamma-dependent manner in a mouse model of asthma.

BACKGROUND: Intrapulmonary administration of IL-12 has been shown to inhibit the number of eosinophils in lung murine models of asthma, but the precise mechanism of this inhibition has not been reported. The purpose of this study was to examine whether IL-12 treatment inhibits bone marrow eosinophilopoiesis, and to elucidate the role of IFN-gamma in this process. OBJECTIVE: To elucidate the in vivo and in vitro effects of IL-12 on eosinophil differentiation from murine bone marrow (BM) stem cells, and to examine the mechanistic role of IFN-gamma in this process. METHODS: Allergen-sensitized BALB/c mice were administered low doses of intranasal IL-12 at the time of allergen challenge, and the number of eosinophils in BM was determined 3 days later. The direct actions of IL-12 on eosinophil differentiation from BM cells were determined in vitro. The mechanistic role of IFN-gamma was assessed by measuring IFN-gamma induction by IL-12 in BM cell cultures, and through the use of IFN-gamma KO mice. RESULTS: Treatment of allergic mice with intrapulmonary IL-12 (1 ng or 10 ng) reduced eosinophils in BM by 43%. Culture of BM cells from allergen-sensitized mice with IL-3 + IL-5 induced eosinophil differentiation in vitro. Addition of IL-12 to these cultures inhibited eosinophil differentiation, with maximal inhibition (45%) occurring at 10 ng/mL IL-12 concentration. IL-12 induced IFN-gamma production from BM cultures, and failed to inhibit eosinophil differentiation in IFN-gamma-knockout mice, indicating a critical mechanistic role for IFN-gamma. CONCLUSION: This study demonstrates that IL-12 selectively inhibits BM eosinophilopoiesis, and that this effect is mediated by IFN-gamma. Intrapulmonary IL-12 has suppressive effects on BM eosinophilopoiesis that may represent a novel mechanism contributing to the anti-eosinophilic effects of IL-12 in allergic airway disease.

Animals↗
PubMed2001

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils.

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.

ADP Ribose Transferases↗
PubMed2000

The differential role of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase in eosinophil functions.

The activation of eosinophils by cytokines is a major event in the pathogenesis of allergic diseases. We have investigated the activation of mitogen-activated protein (MAP) kinases and their functional relevance in eosinophil differentiation, survival, degranulation, and cytokine production. IL-5 induced phosphorylation and activation of extracellular signal-regulated kinases (ERK) and p38 MAP kinases in eosinophils. PD98059, a MAP/ERK kinase inhibitor, blocked phosphorylation of ERK1/2 in a dose-dependent manner. SB202190, a p38 inhibitor, blocked p38-dependent phosphorylation of activating transcription factor-2. To study the importance of the MAP kinases on eosinophil differentiation, we cultured mouse bone marrow cells with IL-3 and IL-5 in the presence of the inhibitors. SB202190 dramatically inhibited eosinophil differentiation by 71%. PD98059 was less potent and reduced eosinophil differentiation by 28%. Both inhibitors marginally inhibited eosinophil survival only at the highest doses. Prolonged incubation of eosinophils with IL-5 induced significant eosinophil-derived neurotoxin release. Both PD98059 and SB202190 nearly completely inhibited (87% and 100% inhibition, respectively) IL-5-stimulated eosinophil-derived neurotoxin release in a dose-dependent manner. Next, we examined the effect of the MAP kinase inhibitors on eosinophil production of the cytokine macrophage-inflammatory protein (MIP)-1alpha. PD98059 blocked C5a- but not ionomycin-induced MIP-1alpha production (59% inhibition at 50 microM concentration). In contrast, SB202190 nearly completely inhibited (99%) C5a-induced MIP-1alpha production. Further, it blocked ionomycin-stimulated production by 66%. Our results suggest that both p38 and ERK1/2 MAP kinases play an important role in eosinophil differentiation, cytokine production, and degranulation. The p38 MAP kinase plays a greater role than ERK1/2 in eosinophil differentiation and cytokine production.

Animals↗
PubMed2000

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases.

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3 and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2000

Rheumatology telephone helplines: an activity analysis. South and West of England Rheumatology Consortium.

BACKGROUND: Anecdotal evidence suggests that the services offered by rheumatology telephone helplines in the UK vary widely between NHS Trusts because of the lack of national or European guidelines. OBJECTIVE: To conduct an activity analysis of six NHS Trust rheumatology telephone helplines in the south and west of England. METHODS: Serial data were collected on the first 100 calls received on or after 1 January 1999 by six rheumatology helplines in the south and west of England. Background information was gathered on the management, availability, setting and purpose of each helpline. Data on the time taken to manage these calls and patient satisfaction were not collected. RESULTS: Patients with rheumatoid arthritis were the major users and no significant differences were found in the outcome of their calls between centres, but wide variations were revealed in the management of the helplines, the populations they serve and the services they offer. CONCLUSION: The rheumatology helpline services in six NHS Trusts in the south and west of England were shown to be the same in name only. They lacked uniformity in the delivery of care and accessibility to relevant patient groups. The geographical variation in service delivery may result in patient dissatisfaction and confusion if a number of hospitals are attended over the course of a patient's chronic disease. Further research is required to identify the helpline needs of the broader rheumatology population, patient satisfaction, outcomes and system costs, and to progress towards the development of national and European guidelines.

Aged↗
PubMed1999

A novel Lyn-binding peptide inhibitor blocks eosinophil differentiation, survival, and airway eosinophilic inflammation.

Receptor antagonists block all receptor-coupled signaling pathways indiscriminately. We introduce a novel class of peptide inhibitors that is designed to block a specific signal from a receptor while keeping other signals intact. This concept was tested in the model of IL-5 signaling via Lyn kinase. We have previously mapped the Lyn-binding site of the IL-5/GM-CSF receptor common beta (beta c) subunit. In the present study, we designed a peptide inhibitor using the Lyn-binding sequence. The peptide was N-stearated to enable cellular internalization. The stearated peptide blocked the binding of Lyn to the beta c receptor and the activation of Lyn. The lipopeptide did not affect the activation of Janus kinase 2 or its association with beta c. The inhibitor blocked the Lyn-dependent functions of IL-5 in vitro (e.g., eosinophil differentiation from stem cells and eosinophil survival). It did not affect eosinophil degranulation. When applied in vivo, the Lyn-binding peptide significantly inhibited airway eosinophil influx in a mouse model of asthma. The lipopeptide had no effect on basophil histamine release or on the proliferation of B cells and T cells. To our knowledge, this is the first report on an inhibitor of IL-5 that blocks eosinophil differentiation, survival, and airway eosinophilic inflammation. This novel strategy to develop peptide inhibitors can be applied to other receptors.

Amino Acid Sequence↗
PubMed1999

The mapping of the Lyn kinase binding site of the common beta subunit of IL-3/granulocyte-macrophage colony-stimulating factor/IL-5 receptor.

It has been shown that a membrane-proximal region within common beta (betac) receptor of IL-3/granulocyte-macrophage CSF/IL-5 (amino acids 450-517) is important for Lyn binding. We have shown previously that Lyn kinase is physically associated with the IL-5R betac subunit in unstimulated cells. The result suggests that this association involves binding modules that are not activation or phosphorylation dependent. The objective of this study was to map the exact Lyn binding site on betac. Using overlapping and/or sequential peptides derived from betac 450-517, we narrowed down the Lyn binding site to nine amino acid residues, betac 457-465. The P-->A mutation in this region abrogated the binding to Lyn, indicating a critical role of proline residues. We created a cell-permeable Lyn-binding peptide by N-stearation. This cell-permeable peptide blocked the association of Lyn, but not Jak2 with betac in situ. We also investigated the betac binding site of Lyn kinase. Our results suggest that the N-terminal unique domain of Lyn kinase is important for binding to betac receptor. To our knowledge, this is the first molecular identification of the Lyn binding site of betac receptor. This finding may help develop specific inhibitors of Lyn-coupled signaling pathways.

Amino Acid Sequence↗
PubMed1998

Lyn, Jak2, and Raf-1 kinases are critical for the antiapoptotic effect of interleukin 5, whereas only Raf-1 kinase is essential for eosinophil activation and degranulation.

Interleukin (IL)-5 has been shown to activate many signaling molecules in eosinophils, but their functional relevance remains unknown. We have examined the functional relevance of Lyn, Jak2, and Raf-1 kinases in eosinophil survival, upregulation of adhesion molecules and degranulation. To this goal we used Lyn and Raf-1 antisense (AS) oligodeoxynucleotides (ODN) to inhibit the expression of these proteins and tyrphostin AG490 to specifically block the activation of Jak2. We have demonstrated that all three kinases are important for IL-5- induced suppression of eosinophil apoptosis. However, Lyn and Jak2 tyrosine kinases are not important for the upregulation of CD11b and the secretion of eosinophil cationic protein. In contrast, Raf-1 kinase is critical for both these functions. This is the first identification of specific signaling molecules responsible for three important functions of eosinophils. We have established a central role for Raf-1 kinase in regulating eosinophil survival, expression of beta2 integrins and degranulation. Further, there appears to be a dissociation between two receptor-associated tyrosine kinases, i.e., Lyn and Jak2, and the activation of Raf-1 kinase. The delineation of the functional relevance of signaling molecules will help design therapeutic approaches targeting specific eosinophil function.

Apoptosis↗
PubMed1997

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody.

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC), a CC chemokine, is chemotactic for cells that typically infiltrate the late-phase allergic reaction. We developed a mouse model of airway inflammation to study the role of MCP-3/FIC. The immunization of mice with OVA resulted in Ag-specific IgE Ab production and the expression of mRNA for IL-4 in the lung tissue. Two weeks after immunization mice were challenged with the allergen by inhalation. Lungs were lavaged, and the tissue was examined at 2 or 24 h. Allergen challenge resulted in the increased recovery of leukocytes in the lavage fluid, but saline challenge did not. There was a significant increase in eosinophils (29 +/- 8% vs 1.2 +/- 0.2%) and lymphocytes (25 +/- 4% vs 5 +/- 2%) in the bronchoaveolar lavage fluid. Histologic examination of the lung demonstrated intense airway inflammation following OVA challenge. The expression of MCP-3/FIC and other CC chemokines (MCP-1, macrophage inflammatory protein-1alpha, and RANTES) was investigated by reverse transcription-PCR followed by densitometric analyses. The allergen challenge up-regulated the expression of mRNA for MCP-1, MCP-3/FIC, and macrophage inflammatory protein-1alpha at 2 and/or 24 h. Immunocytochemical staining for MCP-3/FIC showed that the allergen challenge induced the expression of MCP-3/FIC predominantly in the airway epithelium. Pretreatment of mice with an anti-MCP-3/FIC Ab significantly inhibited the OVA-induced airway inflammation and the bronchoalveolar lavage eosinophilia (8 +/- 2% vs 46 +/- 11% after control Ab, p < 0.03). We conclude that MCP-3/FIC plays a significant role in the allergen-induced eosinophilic inflammation of the airways.

Animals↗
PubMed1996

Increased MCP-1, RANTES, and MIP-1alpha in bronchoalveolar lavage fluid of allergic asthmatic patients.

Chemokines are cytokines that induce chemotaxis of inflammatory cells. We studied the presence of chemokines in bronchoalveolar lavage fluid (BALF) obtained from nine allergic asthmatic patients and six nonsmoking normal individuals. The cells were pelleted, and ribonucleic acid (RNA) was extracted by using RNAzol B. BALF was assayed for monocyte chemoattractant protein-1 (MCP-1), regulated upon activation in normal T cells, expressed, probably secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha) and interleukin-8 (IL-8) by enzyme-linked immunosorbent assay (ELISA). The levels of MCP-1, RANTES, and MIP-1alpha were significantly higher in the asthma patients than in the control subjects (p<0.04). The concentrations of RANTES and MCP-1 correlated with the lymphocyte count in the BAL specimens (r = 0.61 and 0.68, respectively). BALF showed eosinophil chemotactic activity in vitro that was blocked by anti-RANTES and anti-MCP-3 antibodies. The total cellular RNA was reverse-transcribed and the complementary deoxyribonucleic acid (cDNA) was amplified with the polymerase chain reaction (PCR) for MCP-1, MCP-3, RANTES, MIP-1alpha, IL-8, and beta-actin. We found that messenger ribonucleic acids (mRNAs) for MCP-1, MCP-3, RANTES, MIP-1alpha, and IL-8 were produced by BAL cells from most asthmatic and normal subjects. We conclude that chemokines are produced in the airways, and that an increased recovery of MCP-1, RANTES, and MIP-1alpha is observed in allergic asthmatic patients.

Adolescent↗
PubMed1995

The activation of the Jak-STAT 1 signaling pathway by IL-5 in eosinophils.

The intracellular signal transduction of IL-5 in eosinophils is unknown. The objective of this study was to investigate the involvement of the newly discovered Jak-STAT pathway in the IL-5 signal transduction mechanism. Eosinophils were purified from peripheral blood by discontinuous Percoll gradients and stimulated with IL-5. The involvement of Jak 2 was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. The activation of Jak 2 was studied by autophosphorylation of the immunoprecipitated kinase. Jak 2 was tyrosine phosphorylated within 1 to 3 min after stimulation of eosinophils with IL-5. Further, the immunoprecipitated Jak 2 obtained from IL-5-stimulated cells underwent autophosphorylation. Jak 2 coprecipitated with the beta-subunit of the IL-5 receptor, suggesting a physical association of the kinase with the receptor. The nuclear factor STAT-1 (p91) was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. STAT-1 was tyrosine phosphorylated within 15 min of IL-5 stimulation. The presence of STAT-1 in the nuclear extract was studied by electrophoretic mobility shift assay. IL-5 induced two proteins that bound to the gamma-activating sequence. In the presence of an anti-STAT-1 Ab, the band was supershifted. Thus, we demonstrated that IL-5 activated the Jak 2-STAT 1 signaling pathway in eosinophils. We speculate that the Jak 2-STAT 1 pathway may be involved in the activation of IL-5-inducible genes in eosinophils.

Base Sequence↗
PubMed1994

Monocyte chemotactic protein-2, monocyte chemotactic protein-3, and fibroblast-induced cytokine. Three new chemokines induce chemotaxis and activation of basophils.

Cytokine-dependent mediator release from basophils and mast cells may play an important role in the pathogenesis of allergic and inflammatory conditions. Many C-C chemokines have been found to activate basophils and mast cells. We investigated the effect of three newly identified C-C chemokines, monocyte chemotactic protein-2 and -3 (MCP-2, MCP-3) and fibroblast-induced cytokine (FIC) on basophils and mast cells. We found that all three cytokines induced histamine secretion from basophils in a dose-dependent manner. The secretion of histamine was a Ca(2+)-dependent process. MCP-3 was the most potent activator of basophils. MCP-3 and FIC activated basophils from all study subjects, whereas the histamine release by MCP-2 was donor-dependent. The histamine-releasing activity of MCP-2, MCP-3, and FIC was compared with that of MCP-1, RANTES, and macrophage inflammatory protein-1 alpha using basophils from 10 donors. MCP-1 was the most potent among all the C-C chemokines. However, MCP-3 was nearly as potent. MCP-2, MCP-3, and FIC induced significant chemotaxis of basophils. None of the cytokines activated mouse peritoneal mast cells. The synthesis of mRNA for MCP-3 was investigated by reverse-transcription PCR using allergen-stimulated PBMC and bronchoalveolar lavage cells. Both MNC and bronchoalveolar lavage cells expressed mRNA for MCP-3. The results of this study indicate that MCP-2, MCP-3, and FIC are novel histamine-releasing factors.

Allergens↗
PubMed1994

Transforming growth factor beta abrogates the effects of hematopoietins on eosinophils and induces their apoptosis.

Hematopoietins, interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) have previously been shown to prolong eosinophil survival and abrogate apoptosis. The objective of this study was to investigate the effect of transforming growth factor beta (TGF-beta) on eosinophil survival and apoptosis. Eosinophils from peripheral blood of mildly eosinophilic donors were isolated to > 97% purity using discontinuous Percoll density gradient. Eosinophils were cultured with hematopoietins with or without TGF-beta for 4 d and their viability was assessed. We confirmed previous observations that hematopoietins prolonged eosinophil survival and inhibited apoptosis. TGF-beta at concentrations > or = 10(-12) M abrogated the survival-prolonging effects of hematopoietins in a dose-dependent manner and induced apoptosis as determined by DNA fragmentation in agarose gels. The effect of TGF-beta was blocked by an anti-TGF-beta antibody. The anti-TGF-beta antibody also prolonged eosinophil survival on its own. The culture of eosinophils with IL-3 and GM-CSF stimulated the synthesis of GM-CSF and IL-5, respectively, suggesting an autocrine mechanism of growth factor production. TGF-beta inhibited the synthesis of GM-CSF and IL-5 by eosinophils. TGF-beta did not have any effect on the expression of GM-CSF receptors on eosinophils. We also studied the effect of TGF-beta on eosinophil function and found that TGF-beta inhibited the release of eosinophil peroxidase. Thus, TGF-beta seems to inhibit eosinophil survival and function. The inhibition of endogenous synthesis of hematopoietins may be one mechanism by which TGF-beta blocks eosinophil survival and induces apoptosis.

Antibodies, Monoclonal↗
PubMed1993

RANTES is a chemotactic and activating factor for human eosinophils.

RANTES is a member of the 8-kDa cytokine family that has been shown to possess chemotactic activity for monocytes and CD4 T cells. In this study, we investigated whether RANTES could affect eosinophil chemotaxis and function. Peripheral blood eosinophils from blood donors were isolated on Percoll gradients to > 98% purity and then used for chemotaxis, flow cytometry, eosinophil cationic protein release assay, and survival assay. We found that RANTES is chemotactic for eosinophils at 10(-9) to 10(-8) M concentrations. RANTES elicited 65% of the chemotactic response to 10(-7) M platelet-activating factor in all experiments. The mechanism of chemotaxis was investigated by studying the expression of adhesion molecules on eosinophils by flow cytometry. We found that RANTES up-regulated the expression of CD11b/CD18 on eosinophils in a dose-dependent manner. In another set of experiments, purified eosinophils incubated with various concentrations of RANTES released eosinophil cationic protein as measured by a RIA. We also investigated the effect of RANTES on eosinophil density. Leukocytes were incubated in the presence or absence of RANTES, and the distribution of eosinophils on discontinuous Percoll gradients was then examined. We found that eosinophils became hypodense (< 1.085) when incubated in RANTES. However, unlike IL-3, RANTES did not affect the survival of eosinophils in a 4-day culture system. Thus, we established that RANTES is a chemotactic and activating factor for eosinophils.

Chemokine CCL5↗