Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells

The role of cyclic AMP in the stimulation of corticotropin (ACTH) release by corticotropin-releasing factor (CRF), angiotensin II (AII), vasopressin (VP), and norepinephrine (NE) was examined in cultured rat anterior pituitary cells. Synthetic CRF rapidly stimulated cyclic AMP production, from 4- to 6-fold in 3 min to a maximum of 10- to 15-fold at 30 min. Stimulation of ACTH release by increasing concentrations of CRF was accompanied by a parallel increase in cyclic AMP formation, with ED50 values of 0.5 and 1.3 nM CRF for ACTH and cyclic AMP, respectively. A good correlation between cyclic AMP formation and ACTH release was also found when pituitary cells were incubated with the synthetic CRF(15-41) fragment, which displayed full agonist activity on both cyclic AMP and ACTH release with about 0.1% of the potency of the intact peptide. In contrast, the CRF(21-41) and CRF(36-41) fragments were completely inactive. The other regulators were less effective stimuli of ACTH release and caused either no change in cyclic AMP (AII and VP) or a 50% decrease in cyclic AMP (NE). Addition of the phosphodiesterase inhibitor, methylisobutylxanthine, increased the sensitivity of the ACTH response to CRF but did not change the responses to AII, VP, and NE. In pituitary membranes, adenylate cyclase activity was stimulated by CRF in a dose-dependent manner with ED50 of 0.28 nM, indicating that the CRF-induced elevation of cyclic AMP production in intact pituitary cells is due to increased cyclic AMP biosynthesis. The intermediate role of cyclic AMP in the stimulation of ACTH release by CRF was further indicated by the dose-related increase in cyclic AMP-dependent protein kinase activity in pituitary cells stimulated by CRF with ED50 of 1.1 nM. These data demonstrate that the action of CRF on ACTH release is mediated by the adenylate cyclase-protein kinase pathway and that the sequence requirement for bioactivity includes the COOH-terminal 27 amino acid residues of the molecule. The other recognized regulators of ACTH release are less effective stimuli than CRF and do not exert their actions on the corticotroph through cyclic AMP-dependent mechanisms.     

p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells.

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.     

Effect of azacytidine in the release of leukemia inhibitory factor,oncostatin m,interleukin (IL)-6, and IL-11 by mononuclear cells of patients with refractory anemia

5-azacytidine (AZA) yields hematologic improvement in patients with myelodysplastic syndromes (MDS). Ineffective hemopoiesis in MDS produce the paradox of high intramedullary cellularity with peripheral cytopenias. Leukemia inhibitory factor (LIF), oncostatin M (OSM), interleukin (IL)-6, and IL-11 regulate hemopoiesis and LIF, OSM, and IL-6 also inhibit the proliferation of myeloid leukemic cell lines through the signal-transducing subunit gp130. These IL-6-type cytokines were measured by enzyme-linked immunosorbent assay in cell culture supernatants (SN) obtained from peripheral blood mononuclear cells (MNC) and monocyte-depleted MNC of patients with refractory anemia (RA; n=12) and healthy individuals (n=10). AZA down-regulated OSM, IL-6, and IL-11 release by MNC of patients but not by MNC from healthy individuals. Patient's SN had significantly lower concentrations of LIF, OSM, and IL-11 than SN of normal subjects. When monocyte-depleted MNC of patients were stimulated with phytohemagglutinin a significant increment in OSM levels was observed. In contrast, monocyte depletion in healthy subjects did not cause any significant change in OSM values. We conclude that: (a) AZA inhibits the release of OSM, IL-6, and IL-11 exclusively in RA-diseased MNC, (b) Patient's MNC release subnormal amounts of LIF, OSM, and IL-11, and (c) RA-derived monocytes probably down-regulate OSM release by phytohemagglutinin-activated MNC.     

Regulation of BSF-2/IL-6 production by human mononuclear cells. Macrophage-dependent synthesis of BSF-2/IL-6 by T cells

Regulation of BSF-2/IL-6 production in peripheral mononuclear cells (MNC) was studied. BSF-2 mRNA expression in mitogen-stimulated MNC showed a biphasic response, the first peak around 4 h and the second peak around 48 h. This was caused by different kinetics of BSF-2 mRNA expression in distinct subpopulations of MNC; M phi expressed BSF-2 mRNA at 5 h in the absence of any stimulation, and mitogen-stimulated T cells and B cells expressed BSF-2 mRNA 48 h after stimulation. Immunohistochemical staining of the cells with anti-BSF-2 antibody demonstrated that macrophages, T cells and B cells could produce BSF-2. T cells in peripheral MNC produced BSF-2 in the presence of M phi. The requirement of macrophage for BSF-2 production in T cells could be replaced by TPA but not by IL-1 or BSF-2.     

IL-12 elispot assays to detect and enumerate IL-12 secreting cells

The cytokine IL-12 promotes Th(1)type immune responses and plays a key role in immune regulation. The complex nature of IL-12 hampered its detection without use of stimulants that might give less relevant information. To detect circulating IL-12 p40, we developed enzyme-linked immunospot (ELISPOT) assays that allow enumeration of IL-12 p40 secreting cells without prior in vitro stimulation of the cells. In parallel, intracellular staining of IL-12 p40 by flow cytometry was performed to compare the two methods. IL-12 p40 secreting cells were detected in healthy subjects at a mean number of 103+/-155 per 10(5)blood mononuclear cells (MNC). Numbers of IL-12 p40 secreting blood MNC correlated with IL-12 p40 positive blood MNC detected by flow cytometry. Bacterial endotoxins and the inflammatory cytokines TNF-alpha and IFN-gamma control IL-12 production by antigen presenting cells. Utilizing IL-12 p40 ELISPOT assays, we could confirm occurrence of elevated numbers of IL-12 p40 secreting blood MNC after stimulation with TNF-alpha, IFN-gamma, LPS, LPS+TNF-alpha or LPS+IFN-gamma, compared to cultures without stimulant. Due to its central role in inflammation and autoimmunity, IL-12 is an attractive target for immunotherapy. IL-12 p40 ELISPOT assays represent a sensitive, specific and reliable tool for investigating the role of IL-12 in both health and disease.     

Distinctive features in the production of IL-6 by human T cells

The production of IL-6 was studied in purified populations of human peripheral blood T cells and monocytes. IL-6 could be elicited from both T cells and thymocytes by a variety of polyclonal stimuli. The expression and production of IL-6 in T cells varied in several ways from that of monocytes: (1) under maximal stimulation, T cells secreted 10-fold less IL-6 than did monocytes; (2) by Western blot, the molecular isoforms produced by T cells were distinct from those produced by monocytes, with at least some isoforms being unique to each cell type; (3) the kinetics of IL-6 expression and production was much slower in T cells than in monocytes; (4) the frequency of labeled cells detected by in situ hybridization was 4% for T cells and 34% for monocytes. Our data indicate that there is considerable diversification among cell types in terms of regulation of IL-6 production and possibly in post-translational modifications. This raises the possibility that the IL-6 produced by different cell types may have varying effects depending on the form produced and the timing of synthesis.     

Independent regulation of B-cell inducing factor and IL-2 production by T lymphocytes, and direct and indirect promotion of immunoglobulin secretion by glucocorticosteroid

The conditions for induction of B-cell inducing factor (BIF) by human peripheral blood T cells was investigated. BIF was assayed by induction of immunoglobulin secreting cells (ISC) in peripheral blood B (non-T) cells stimulated with Staphylococcus aureus bacteria strain Cowan I (Sac), and in the IgM cell line SKW6.4. Maximum BIF production occurred with high concentrations of the T-cell mitogens phytohemagglutinin, concanavalin A, and PWM. Dexamethasone (Dex) also induced BIF production in T cells at 10(-5) to 10(-7) M. At 10(-5) and 10(-6) M Dex, the T-cell supernatants had to be dialyzed before testing because Dex alone stimulated variable levels of ISC in both test B-cell assays. Dex did not enhance BIF production by T cells that were optimally stimulated by lectin. BIF levels were maximum by Day 2 of T-cell cultures and remained high at Days 3 and 4. In contrast, IL-2 reached a peak at Day 1 and declined drastically by Day 4. We previously showed that IL-2 at less than 100 U/ml did not induce ISC in B cells and did not alter ISC induction by BIF. Dex did not induce IL-2 production and inhibited IL-2 production induced by Con A, in contrast to the promoting effects of Dex on BIF production, providing further evidence for the independence of BIF and IL-2 production and B-cell stimulation.     

Effect of L-phenylalanine methyl ester on the colony formation of hematopoietic progenitor cells from human bone marrow

We have previously shown that L-phenylalanine methyl ester (PME) is capable of removing monocytes and enhancing the growth of hematopoietic colonies from human peripheral blood (PB) mononuclear cells (MNC). In the present study, we further compared the effect of PME on the colony formation of bone marrow (BM) and PB. Low density (less than or equal to 1.077 g/ml) MNC were obtained by Ficoll-diatrizoate density gradient centrifugation. Granulocyte/macrophage colony-forming units (CFU-gm) and erythroid burst-forming units (BFU-e) were cultured in agarose with conditioned media (CM) and/or interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage-CSF (GM-CSF). Treatment of BM MNC with 5 mM PME for 15 min at room temperature yielded a nucleated cell recovery of 44.8 +/- 5.0% (mean +/- SE; N = 8). CFU-gm were enriched 2.7-fold (range 2.0 to 4.8). Using CM or CM supplemented with G-CSF or GM-CSF has minimal effect on the enrichment. Leukocyte differentials revealed that 94.3 +/- 3.05% of the monocytes, as well as 91.2 +/- 1.60% of the cells in the neutrophilic maturation series were removed by PME. Incubation for 40 min in PME abolished CFU-gm formation. BFU-e were not enriched by the PME treatment. In contrast, 40 min incubation of PB MNC produced higher enrichment of CFU-gm than that obtained from 15 min of treatment, although lower cell recovery was obtained with the longer treatment time. In conclusion, we have demonstrated that phagocytic cells can be removed from BM or PB MNC by PME treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.     

Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF- proportional, variant, IL-6, PGE(2) and LTB(4) by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF- proportional, variant, IL-6 and PGE(2) production but had no effect on LTB(4) production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the K(d) or receptor number.     

Generation of killer activity by interleukin-12 of mononuclear cells in malignant pleural effusions due to lung cancer

 In the present study, we examined the ability of interleukin (IL)-12 to generate an antitumor effect in the tumor-growing site. Mononuclear cells (MNC) were obtained from 12 malignant pleural effusions due to lung cancer in the tumor-growing site. Non-major-histocompatibility-complex-restricted killer activity, examined by 4-h ⁵¹Cr release assay against Daudi lymphoma cells as well as various lung cancer cell lines (H69 and PC-9), and in vitro production of interferon γ (IFNγ), measured by enzyme immunoassay, were investigated as mediators of antitumor effects of host cells activated by IL-12. IL-12 induced killer activity of MNC in pleural effusions (pleural MNC) dose-dependently. Moreover, pleural MNC produced a signficant amount of IFNγ in response to IL-12. The killer activities of IL-12-activated blood MNC were higher than those of pleural MNC. The supernatants of pleural effusions of these untreated patients suppressed killer induction by IL-12 of blood MNC of healthy volunteers. These observations suggest that MNC present at the site of growing tumors may act as effector cells against lung cancer in the presence of IL-12. Received: 31 December 1996 / Accepted: 10 September 1997     

The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.     

Interleukin (IL-)15 has less activity than IL-2 to promote type 2 cytokine predominance in tumour-associated mononuclear cells from lung cancer patients

Deviation of type 1/type 2 cytokine balance to type 2 predominance may contribute to tumour progression. We investigated effect of interleukin (IL-)15 on modulation of type 1/type 2 balance in addition to non-major histo-compatibility complex (MHC)-restricted killer induction in the tumour-growing site. IL-15 induced significant killer activity in mononuclear cells (MNC) in malignant pleural effusion as well as those in peripheral blood. Pleural MNC produced more IFN-gamma (type 1 cytokine) by incubation with IL-15 or IL-2 than blood MNC. Moreover, IL-4 and IL-5 (type 2 cytokines) production by pleural MNC were observed only by incubation with IL-2, but not with IL-15. These observations suggest that IL-15 has a potent activity to restore type 1/type 2 balance in addition to killer induction in tumour-growing site.     

Sustained engraftment of mice transplanted with IL-1-primed blood-derived stem cells.

IL-1 is considered the primary mediator of the acute phase response. One of the characteristic manifestations of this response is early neutrophilia that is probably caused by release of mature neutrophils from the bone marrow into the peripheral blood. In the present study, we assessed whether IL-1 had a similar releasing effect on the number of circulating progenitor cells and stem cells. Female BALB/c mice were injected i.p. with increasing (0.1-1.0 micrograms/mouse) concentrations of rhu-IL-1 alpha. IL-1 injection resulted in a marked dose-dependent increase in the number of polymorphonuclear neutrophils, granulocyte-macrophage colony-forming units (CFU-GM), and cells forming spleen colonies (CFU-S day 8 and day 12). The maximal increase was found at 4 to 8 h after injection of 1 micrograms IL-1 per mouse, yielding a mean fivefold elevation in neutrophil count, and a mean 30-fold and 10-fold increase in the number of circulating CFU-GM and CFU-S, respectively. In a subsequent series of experiments, lethally irradiated (8.5 Gy) female recipient animals were transplanted with 5 x 10(5) blood mononuclear cells derived from male IL-1-treated animals. Long-term survival was obtained in 68% of mice transplanted with peripheral blood cells derived from donor animals at 6 h after a single injection of 1 micrograms IL-1. The mean number of circulating CFU-GM in these donor animals was 557/ml blood. At 6 mo after transplantation, greater than 95% of the bone marrow cells were of male origin, as determined using in situ hybridization with a Y-chromosome specific probe. In contrast, long-term survival was reached in less than 10% of mice transplanted with an equal number of blood cells derived from saline-treated controls or donor animals treated with a dose of 0.1 micrograms IL-1. These results indicate that a single injection of IL-1 induces a shift of hematopoietic progenitor cells and marrow repopulating cells into peripheral blood and that these cells can be used to rescue and permanently repopulate the bone marrow of lethally irradiated recipients.     

Influence of sex hormones on Coxsackie B-3 virus infection in Balb/c mice

Background and “spontaneous” proliferation are terms often used for the proliferative activity normally exhibited by peripheral blood mononuclear cells (MNC) in vitro. In this report, we show that Interleukin-2 (IL-2) added to unfractionated MNC but not to isolated T or non-T cells significantly increased their proliferative activity. The cells responding to IL-2 stimulation from MNC were OKT3 positive lymphocytes. In addition, treatment of MNC with either a monoclonal anti-HLA-DR antibody (in the absence of C′) or Cyclosporin-A strongly suppressed the “background” whereas treatment of MNC with the 3A1 monoclonal anti-human T cell antibody did not modify “spontaneous” proliferation of these cells. IL-2 could not restore or increase the proliferative activity of MNC exposed to the anti-HLA-DR antibody or Cyclosporin-A while the T cell growth factor significantly enhanced proliferation of MNC cultured in the presence of the OKT4 antibody. Taken together these results strongly suggest that IL-2 responding T cells from MNC become sensitive to IL-2 by interacting with HLA-DR antigens on B lymphocytes and/or monocytes contained in MNC (resting T cells are Dr^?). By a similar mechanism we have previously shown that T cells acquire responsiveness to IL-2 in the autologous mixed lymphocyte reaction (AMLR). Since all the cells that participate in AMLR are present in MNC, we postulate that a “mini” AMLR taking place within MNC may explain the “spontaneous” proliferation of peripheral blood mononuclear cells.     

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.     

Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage-dependent regulation of functional receptors

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.