Regulatory effect of cytokines on eosinophil degranulation

We tested the effects of different cytokines on IgA- and IgG-induced eosinophil degranulation in vitro to determine the potential interaction between eosinophils and mononuclear cells. Purified normodense eosinophils were incubated with cytokines (including rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, IFN-gamma, granulocyte-macrophage CSF stimulating factor (GM-CSF), and TNF) for 1 to 3 h after which Ig-coupled Sepharose 4B beads were added as targets and the mixtures were incubated with the eosinophils at 37 degrees C for 4 h. The Ig used were secretory IgA (sIgA), serum IgA and IgG, and myeloma IgA and IgG. The release of eosinophil-derived neurotoxin (EDN) was measured by RIA as an index of degranulation. rIL-5 was the most potent enhancer of Ig-induced degranulation and increased EDN release by 48% for sIgA and 136% for IgG. The effect of rIL-5 appeared as quickly as 15 min after incubation of eosinophils, sIgA beads and IL-5. GM-CSF and rIL-3 also enhanced Ig-induced EDN release but less potently than rIL-5. GM-CSF and rIL-5 by themselves induced a small but significant release of EDN from eosinophils in the absence of Ig-coated beads; rIL-3 did not. However, IFN-gamma suppressed sIgA-induced EDN release by 23%. The other cytokines did not have any effect on eosinophil degranulation. These results suggest that cytokines which induce eosinophil differentiation and proliferation during hematopoiesis also enhance the effector function of mature eosinophils and that IFN-gamma partially down-regulates eosinophil degranulation.     

IgA-induced eosinophil degranulation

Eosinophils play an important role as effector cells in allergic, parasitic, and other conditions. The mechanism(s) by which eosinophils mediate their effector functions was studied by incubation of human normodense eosinophils with Sepharose beads coupled to various Ig isotypes as targets. Controls included eosinophils incubated alone or incubated with uncoated beads, human serum albumin-, or OVA-coated beads. An eosinophil granule protein, the eosinophil-derived neurotoxin (EDN), was measured as an indicator of eosinophil degranulation. Eosinophils released eosinophil-derived neurotoxin when incubated with Sepharose beads coupled to Ig of the IgG or IgA isotypes, as well as IgA-Fc fragments. Mixtures of IgG and IgA on beads did not act synergistically. Secretory IgA (sIgA) provided the most potent signal for eosinophil degranulation and was two to three times more potent than IgG. Furthermore, 2 to 17% of the normodense eosinophils bound to IgG- or IgA-coated beads, whereas 24 to 27% of the eosinophils bound to sIgA-coated beads. Thus, sIgA may be the principal Ig mediating eosinophil effector function at mucosal surfaces in helminth infections and hypersensitivity diseases, especially bronchial asthma.     

Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophil degranulation.

Stimulation of human normodense eosinophils with immobilized secretory IgA (sIgA) or IgG, or with the soluble stimulus, FMLP, triggers the exocytotic release of the granule protein, eosinophil-derived neurotoxin (EDN). In this report, we demonstrate that these stimuli also provoke an increase in phospholipase C-mediated phosphoinositide breakdown in eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) for 2 h irreversibly abolished the increases in phospholipase C activity and EDN release induced by immobilized sIgA or FMLP. In contrast, PTX treatment only transiently inhibited eosinophil activation induced by immobilized IgG. Maximal inhibition of IgG-stimulated phosphoinositide hydrolysis and EDN release occurred after 2 h of PTX pretreatment with PTX, followed by a gradual recovery of cellular responsiveness to immobilized IgG as the duration of PTX pretreatment was extended to 16 h. Activated PTX catalyzed the in vitro ADP-ribosylation of 41- and 44-kDa proteins in eosinophil membranes. A 2-h pretreatment of intact cells with PTX markedly reduced the pools of unmodified 41- and 44-kDa substrates available for subsequent ADP-ribosylation in vitro, suggesting that both proteins were substrates for PTX in intact eosinophils. Continuous exposure of eosinophils to PTX for times ranging from 2 to 15 h resulted in the gradual reappearance of unmodified 44-kDa protein, whereas the levels of unmodified 41-kDa protein were persistently reduced in PTX-treated cells. The time course of the decline and reappearance of unmodified 44-kDa substrate in PTX-treated eosinophils closely paralleled the changes in the responsiveness of these cells to immobilized IgG. These results suggest that the receptors for sIgA, FMLP, or IgG transduce activating signals for eosinophil degranulation through differential coupling to at least two PTX-sensitive G proteins.     

Degranulation of human eosinophils induced by Paragonimus westermani-secreted protease

Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EDN) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Calcium ionophore A23187 calcium-dependent cytolytic degranulation in human eosinophils

The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.     

IL-3 and TNFα increase Thymic Stromal Lymphopoietin Receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation

Background

Thymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses.

Methods

Eosinophil mRNA expression of TSLPR and IL-7R?? was examined by real-time quantitative PCR of human eosinophils treated with TNF?? and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNF?? and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation.

Results

Eosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNF?? and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNF?? and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation.

Conclusions

This study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Prostaglandin E2 potentiates granulocyte-macrophage colony stimulating factor-induced histamine synthesis in bone marrow cells: role of cAMP.

Histamine synthesis in response to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) by murine hematopoietic cells is strikingly potentiated by prostaglandin E2 (PGE2). This synergy is mediated by an increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP), since: (a) exogeneous and endogeneous cAMP generated either by forskolin or IBMX potentiate GM-CSF-induced histamine synthesis, (b) the maximal potentiating effects of PGE2 and cAMP are not cumulative, and (c) GM-CSF together with PGE2 enhances intracellular cAMP content in a bone marrow population enriched for GM-CSF target cells. cAMP and PGE2 enhance histidine decarboxylase activity induced by GM-CSF showing that both factors act on histamine synthesis rather than on its release. Conversely, histamine synthesis promoted by Interleukin 3 (IL-3), the unique cytokine sharing this property with GM-CSF, is not modulated by PGE2 or cAMP, suggesting two distinct mechanisms for the induction of this biological activity in hematopoietic cells.     

Release of granule proteins from eosinophils cultured with IL-5.

Eosinophils isolated from normal individuals were cultured in the presence of human rIL-5 (hrIL-5) for up to 14 days, and the effects of this exposure were determined. First, the hrIL-5-cultured eosinophils were activated and degranulated more readily than freshly isolated eosinophils. For example, eosinophils cultured for 7 days with hrIL-5 released 30 and 10% of granule eosinophil-derived neurotoxin (EDN) when exposed to Sepharose 4B beads coupled to secretory IgA and IgG, respectively, whereas freshly isolated eosinophils released only 19 and 4%, respectively, of their EDN in response to the same stimuli. Degranulation of hrIL-5-cultured eosinophils was not augmented by further exposure to hrIL-5, whereas degranulation of freshly isolated cells to secretory IgA and IgG beads was increased by exposure to hrIL-5. Second, eosinophils cultured with hrIL-5 had prolonged viability in vitro. For example, after four days of culture with 50 U/ml of hrIL-5, 86% of eosinophils were viable compared to 12% in medium alone. Third, hrIL-5-cultured eosinophils became hypodense, and electron microscopy showed that they contained granules with core and matrix lucency and with evidence of granule fusion. Fourth, hrIL-5-cultured eosinophils spontaneously lost 30 to 60% of their EDN, eosinophil cationic protein, and eosinophil peroxidase and about 50% of their eosinophil granule major basic protein content compared to freshly isolated eosinophils, and all four of the granule proteins were released into the culture medium. Fifth, detailed studies of eosinophils cultured in hrIL-5 showed that 89 +/- 10% of the starting quantity of EDN could be recovered at 7 days. Whereas 99 +/- 1% of the EDN at day 0 was cell associated, by 7 days 60 +/- 9% was in the cell supernatants. Thus, hrIL-5 activates eosinophils, increases their viability, decreases their density, and their content of granule proteins and causes release of the granule proteins into culture fluids. The striking loss of granule proteins during culture with hrIL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role.     

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.     

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

Direct effects of prolactin on corticosterone release by zona fasciculata-reticularis cells from male rats

The role of prolactin (PRL) in the male is not fully defined. The aim of this study was to investigate the function and mechanism of PRL on the production of corticosterone by zona fasciculata-reticularis (ZFR) cells in vitro. The ZFR cells were obtained from male rats under normal, hyperprolactinemic, or hypoprolactinemic situation. PRL stimulated the corticosterone release in a dose-dependent pattern in the ZFR cells from normal male rats. The cellular adenosine 3'-5'-cyclic monophosphate (cAMP) concentration positively correlated with PRL concentration in the presence of forskolin or 3-isobutyl-1-methylxanthine (IBMX). PRL enhanced the stimulatory effects of cAMP mimetic reagents, i.e., forskolin, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and IBMX on the release of corticosterone. The adenylate cyclase inhibitor (SQ22536) inhibited the corticosterone release in spite of presence of PRL. Nifedipine (L-type calcium channel blocker) did not inhibit corticosterone release. The hyperprolactinemic condition was actualized by transplantation of donor rat anterior pituitary glands (APs) under kidney capsule. By comparison with the cerebral cortex (CX)-grafted group, AP-graft resulted in an increased release of corticosterone, 3beta-hydroxysteriod dehydrogenase (HSD) activity and cAMP production by ZFR cells. Acute hypoprolactinemic status was induced by bromocriptine for 2 days. The results showed the productions of corticosterone were lower in hypoprolactinemic group than in control group, which were persistent along with different ACTH concentrations. These results suggest that PRL increase the release of corticosterone by ZFR cells via cAMP cascades and 3beta-HSD activity.     

Prostaglandin E2 acts at two distinct pathways of T lymphocyte activation: inhibition of interleukin 2 production and down-regulation of transferrin receptor expression

The mechanism by which prostaglandin E2 (PGE2) inhibits human T lymphocyte activation and proliferation was studied. We analyzed the effect of physiologic concentrations of PGE2 on interleukin 2 (IL 2) production, expression of IL 2 receptor (Tac antigen), and expression of the transferrin receptor after in vitro activation with phytohemagglutinin. PGE2 inhibited T lymphocyte proliferation by 80 to 90% of control values. This was associated with a similar degree of inhibition of IL 2 production while the expression of IL 2 receptor was not affected. This was in marked contrast to the expression of the transferrin receptor, which was inhibited 65% after 72 hr of in vitro activation. The addition of exogenous, purified IL 2 reconstituted lymphocyte proliferation to 50% of control values, but had no effect on transferrin receptor expression. Because PGE2 is known to increase the intracellular concentration of 3',5' cyclic adenosine monophosphate (cAMP), we investigated the effect of another adenylate cyclase activator, i.e., isoproterenol, as well as the effect of extracellular administration of the cAMP derivative dibutyryl cAMP (dBcAMP) on IL 2 production, Tac antigen expression, and transferrin receptor expression. It was demonstrated that isoproterenol, as well as dBcAMP, inhibited transferrin receptor expression on PHA-activated T lymphocytes to the same extent as PGE2, and exogenous IL 2 could not counteract the down-regulation of the receptor expression. In contrast, neither isoproterenol nor dBcAMP had any significant effect on IL 2 receptor expression. Prostaglandin F2 alpha (PGF2 alpha), which has been reported to elevate intracellular cyclic GMP levels, had no effect on lymphocyte activation and proliferation, and did not counteract the PGE2-induced depression in IL 2 production. In contrast to its effect on peripheral blood lymphocytes, PGE2 had no effect on transferrin receptor expression or cell proliferation by IL 2-dependent T cell clones and IL 2-independent T cell lines. These studies demonstrate that PGE2 exerts its inhibitory effects on T cell activation and proliferation via two distinct pathways: inhibition of IL 2 production and inhibition of transferrin receptor expression. The transferrin receptor inhibition is mediated via the cAMP pathway and is IL 2-independent.     

Characterization of the 3',5'-cyclic adenosine monophosphate-mediated regulation of IL2 production by T cells and Jurkat cells.

The effect of cyclic AMP-elevating agents on mitogen-stimulated IL2 production was examined. Prostaglandin E2 (PGE2) inhibited IL2 production by human peripheral blood T cells stimulated with PHA. In contrast, PGE2 did not inhibit PHA-stimulated IL2 production by the human leukemic T cell line. Jurkat, and often slightly enhanced IL2 production by those cells. Other cyclic adenosine monophosphate (cAMP) elevating agents (forskolin, isoproterenol, and the cAMP analogue, dibutyryl cAMP) also inhibited lectin-stimulated IL2 production by T cells, but could not inhibit IL2 production by Jurkat cells. Of the cAMP-elevating agents examined, only cholera toxin (CT) inhibited IL2 production by both Jurkat cells and peripheral blood T cells. Although phorbol myristate acetate (PMA) greatly enhanced PHA-stimulated IL2 production by Jurkat cells. CT remained markedly inhibitory. The combination of PMA and the calcium ionophore, ionomycin, also induced IL2 production by Jurkat cells, and this was similarly suppressed by CT, suggesting that a step after initial second messenger generation was inhibited. A prolonged increase in intracellular cAMP levels was induced by CT in both T cells and Jurkat cells, but the maximal level and the length of elevation achieved in T cells were much less than those observed in Jurkat cells. In contrast, PGE2 caused only a modest and transient increase in intracellular cAMP levels in Jurkat cells compared to that noted with T cells. PGE2 induced a more marked and sustained increase in cAMP levels in Jurkat cells treated with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Moreover, in the presence of IBMX, PGE2 caused a marked inhibition of IL2 production by PHA-stimulated Jurkat cells. Differences in the capacity of PGE2 to induce cAMP could not be explained by disparities in the level of cAMP phosphodiesterase activity as this was comparable in Jurkat cells and in T cells. Thus, these observations indicate that IL2 production by both peripheral T cells and Jurkat cells can be modulated by cAMP-elevating agents. The data suggest that the diminished capacity of PGE2 to inhibit IL2 production by Jurkat cells reflects both a diminished capacity of PGE2 to induce increases in cAMP levels in these cells and an increase in the threshold of cAMP required to inhibit Jurkat cells.     

Delineation of the mechanism of inhibition of human T cell activation by PGE2

The capacity of PGE2 to inhibit human T cell responses was examined by investigating its effect on mitogen-induced IL-2 production and proliferation of highly purified CD4+ T cells. PGE2 inhibited both PHA and anti-CD3 induced proliferation and IL-2 production by an action directly on the responding T cell. Inhibition of IL-2 production reflected decreased accumulation of mRNA for IL-2. A variety of other cAMP elevating agents exerted similar inhibitory effects. Inhibition of proliferation could be overcome by supplemental IL-2, PMA, or the anti-CD28 mAb 9.3. Although PMA and 9.3 markedly increased the amount of IL-2 produced by mitogen-stimulated T cells, the percentage inhibition of IL-2 secretion caused by PGE2 and other cAMP elevating agents remained comparable in these costimulated cultures. Rescue of T cell DNA synthesis by these agents appeared to reflect the finding that, although PGE2 markedly inhibited IL-2 production, the absolute amount of IL-2 produced was increased sufficiently to sustain mitogen-induced proliferation. As anticipated, PGE2, forskolin, and cholera toxin increased T cell cAMP levels. The quantity of cellular cAMP generated in response to PGE2, cholera toxin, and forskolin could be inhibited by PMA or 2',5'-dideoxyadenosine. Using these reagents, the inhibitory effects of PGE2 were found to reflect intracellular cAMP levels, but only within a very narrow range. The results indicate that by elevating cAMP levels, PGE2 inhibits human T cell IL-2 production at a point that is common to both the CD3 and CD28 signaling pathways.     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis

Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.