Modulation of the effects of lung immune response on bone marrow by oral antigen exposure.

Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LTA) single nucleotide polymorphisms: importance in ARDS in septic pediatric critically ill patients.

Accumulating evidence indicates that genetic background influences the outcome of sepsis, which despite medical advances continues to be a major cause of morbidity and mortality. This study aimed to evaluate the influence of SNPs LTA +252A>G, TNF-863C>A and TNF-308G>A on susceptibility to sepsis, acute respiratory distress syndrome (ARDS), septic shock and sepsis mortality. A prospective case-control study was carried out in a Brazilian pediatric intensive care unit and included 490 septic pediatric patients submitted to mechanical ventilation and 610 healthy children. No SNP association was found with respect to sepsis susceptibility. Nevertheless, a haplotype was identified that was protective against sepsis (+252A/-863A/-308G; OR=0.65; p=0.03). We further observed protection against ARDS in TNF-308 GA genotype carriers (OR=0.29; p=0.0006) and -308A allele carriers (OR=0.40; p=0.003). In addition, increased risk for ARDS was detectable with the TNF-863 CA genotype (OR=1.83; p=0.01) and the -863A carrier status (OR=1.82; p=0.01). After stratification according to age, this outcome remained significantly associated with the -308GA genotype in infants. Finally, protection against sepsis-associated mortality was found for the TNF-308 GA genotype (OR=0.22; p=0.04). Overall, our findings document a protective effect of the TNF-308 GA genotype for the ARDS and sepsis mortality outcomes, further providing evidence for an increased risk of ARDS associated with the TNF-863 CA genotype. Trial registration (www.clinicaltrials.gov): NCT00792883.

Evidence for a regulatory role of alpha 4-integrins in the maturation of eosinophils generated from the bone marrow in the presence of dexamethasone.

BACKGROUND: Although eosinophils co-express multiple integrin receptors, the contributions of integrins to eosinophil development have not been explored. We previously described extensive aggregation and cytological immaturity in eosinophils developing in bone-marrow (BM) cultures exposed to dexamethasone. Here we examined the relationship of alpha 4 integrins with these effects of dexamethasone. OBJECTIVES: We evaluated: (a) the effects of exposure to dexamethasone in BM culture on eosinophil expression of alpha 4 integrin receptors and ligands; (b) the contribution of alpha 4 integrins to eosinophil aggregation and maturation. METHODS: Cultures were established with IL-5 (alone or with dexamethasone) for up to 7 days, and eosinophil production, alpha 4 integrin receptor/ligand expression, aggregation and morphology were evaluated before and after targeting alpha 4 integrin-dependent adhesions. Because prostaglandin E2 (PGE2) modifies the effects of dexamethasone on eosinophilopoiesis, PGE2 effects on alpha 4 integrin expression and function were also evaluated. RESULTS: Dexamethasone increased the yield of eosinophils up to day 7. The frequency of eosinophils expressing alpha 4, beta1 and beta 7 integrin receptors at day 7 was also increased by dexamethasone. Eosinophils also expressed the alpha 4 beta 1 ligand, VCAM-1. Dexamethasone increased the expression of alpha 4 integrin and VCAM-1 in aggregates containing eosinophils as early as day 3. PGE2, added up to day 3, modified the effects of dexamethasone to suppress the expression of alpha 4 integrin, decrease aggregation and promote cytological maturation of eosinophils recovered at day 7. Dissociation of immature eosinophils from clusters present at day 3 by reagents targeting alpha 4 or beta1 integrins or VCAM-1 also induced cytological maturation. The concordant effects of targeting alpha 4 integrins with drugs and antibodies support a relationship between alpha 4-mediated aggregation and maturational arrest. CONCLUSIONS: These observations support a novel role for alpha 4 integrin receptors and ligands in eosinophilopoiesis. In addition, increased alpha 4 expression following glucocorticoid exposure may contribute to the retention and accumulation of eosinophils in haemopoietic tissue.

Ectopic lung transplantation induces the accumulation of eosinophil progenitors in the recipients' lungs through an allergen- and interleukin-5-dependent mechanism.

BACKGROUND: Airway challenge of ovalbumin-sensitized mice induces intrapulmonary accumulation of eosinophil progenitors. OBJECTIVE: To evaluate whether allergen-challenged lungs release factors promoting intrapulmonary accumulation of haemopoietic cells, and define the role of allergic lung injury, we developed a transplantation model. METHODS: Lung tissue from allergen-challenged, sensitized donors was ectopically grafted in syngeneic recipients, and haemopoietic progenitors inside the lungs of the recipients were quantified. RESULTS: In BALB/c mice, accumulation of progenitors occurred only when: (a) donors were sensitized and airway challenged with homologous allergen; (b) and recipients were sensitized. Grafts from the appropriate donors released biologically active IL-5, which was effective in sensitized recipients. The effect of the appropriate donor-recipient combination was prevented by neutralizing anti-IL-5 antibody. Grafts from unchallenged, sensitized donors synergized with recombinant IL-5 in sensitized recipients. Unlike BALB/c, grafts from naïve IL-5 transgenic CBA/Ca mice (whose lungs contained a large number of progenitors, independently of sensitization and challenge) were effective in non-transgenic, ovalbumin-sensitized recipients. CONCLUSION: This shows that: (a) intrapulmonary accumulation of progenitors is independent of immunological injury; (b) grafts systemically release IL-5, which is required for progenitor accumulation in the recipients' lungs; (c) and sensitization is required for full responsiveness to IL-5 and for generation of lung-derived signals that synergize with IL-5.

Increased production of tumor necrosis factor-alpha in whole blood cultures from children with primary malnutrition.

Because low tumor necrosis factor-alpha (TNF-alpha) production has been reported in malnourished children, in contrast with high production of TNF-alpha in experimental protein-energy malnutrition, we reevaluated the production of TNF-alpha in whole blood cultures from children with primary malnutrition free from infection, and in healthy sex- and age-matched controls. Mononuclear cells in blood diluted 1:5 in endotoxin-free medium released TNF-alpha for 24 h. Spontaneously released TNF-alpha levels (mean +/- SEM), as measured by enzyme immunoassay in the supernatants of unstimulated 24-h cultures, were 10,941 +/- 2,591 pg/ml in children with malnutrition (N = 11) and 533 +/- 267 pg/ml in controls (N = 18) (P < 0.0001). TNF-alpha production was increased by stimulation with lipopolysaccharide (LPS), with maximal production of 67,341 +/- 16,580 pg/ml TNF-alpha in malnourished children and 25,198 +/- 2,493 pg/ml in controls (P = 0.002). In control subjects, LPS dose-dependently induced TNF-alpha production, with maximal responses obtained at 2000 ng/ml. In contrast, malnourished patients produced significantly more TNF-alpha with 0.02-200 ng/ml LPS, responded maximally at a 10-fold lower LPS concentration (200 ng/ml), and presented high-dose inhibition at 2000 ng/ml. TNF-alpha production a) was significantly influenced by LPS concentration in control subjects, but not in malnourished children, who responded strongly to very low LPS concentrations, and b) presented a significant, negative correlation (r = -0.703, P = 0.023) between spontaneous release and the LPS concentration that elicited maximal responses in malnourished patients. These findings indicate that malnourished children are not deficient in TNF-alpha production, and suggest that their cells are primed for increased TNF-alpha production.

Increased production of tumor necrosis factor-alpha in whole blood cultures from children with primary malnutrition

Because low tumor necrosis factor-alpha (TNF-alpha) production has been reported in malnourished children, in contrast with high production of TNF-alpha in experimental protein-energy malnutrition, we reevaluated the production of TNF-alpha in whole blood cultures from children with primary malnutrition free from infection, and in healthy sex- and age-matched controls. Mononuclear cells in blood diluted 1:5 in endotoxin-free medium released TNF-alpha for 24 h. Spontaneously released TNF-alpha levels (mean ± SEM), as measured by enzyme immunoassay in the supernatants of unstimulated 24-h cultures, were 10,941 ± 2,591 pg/ml in children with malnutrition (N = 11) and 533 ± 267 pg/ml in controls (N = 18) (P < 0.0001). TNF-alpha production was increased by stimulation with lipopolysaccharide (LPS), with maximal production of 67,341 ± 16,580 pg/ml TNF-alpha in malnourished children and 25,198 ± 2,493 pg/ml in controls (P = 0.002). In control subjects, LPS dose-dependently induced TNF-alpha production, with maximal responses obtained at 2000 ng/ml. In contrast, malnourished patients produced significantly more TNF-alpha with 0.02-200 ng/ml LPS, responded maximally at a 10-fold lower LPS concentration (200 ng/ml), and presented high-dose inhibition at 2000 ng/ml. TNF-alpha production a) was significantly influenced by LPS concentration in control subjects, but not in malnourished children, who responded strongly to very low LPS concentrations, and b) presented a significant, negative correlation (r = -0.703, P = 0.023) between spontaneous release and the LPS concentration that elicited maximal responses in malnourished patients. These findings indicate that malnourished children are not deficient in TNF-alpha production, and suggest that their cells are primed for increased TNF-alpha production.

Murine myeloid progenitor responses to GM-CSF and eosinophil precursor responses to IL-5 represent distinct targets for downmodulation by prostaglandin E(2).

1. Because Prostaglandin E(2) (PGE(2)) and dibutiryl cyclic AMP (dbcAMP) modulate the production and effects of haemopoietic cytokines in allergy, we examined their ability to modulate responses of myeloid progenitors to GM-CSF, and of eosinophil precursors to IL-5. 2. The ability of PGE(2), dbcAMP, rolipram, forskolin, dbcGMP and PGD(2), to modulate the responses to GM-CSF and IL-5 in colony formation (progenitor) and eosinophil differentiation (precursor) assays using bone-marrow from nonsensitized or from intranasally-challenged, ovalbumin-sensitized mice of five strains was studied. 3. PGE(2) (10(-7) M) inhibited GM-CSF-stimulated colony formation in bone-marrow from BP-2 mice. This effect was duplicated by dbcAMP (0.3 - 1x10(-6) M), Rolipram (10(-5) M) and forskolin (3x10(-5) M), but not Prostaglandin D(2) (10(-6) M). Inhibition affected similarly all myeloid colony types. Progenitors from sensitized and challenged BP-2 mice were also inhibited by PGE(2) and cyclic AMP. PGE(2) inhibited progenitors from C57BL/10, CBA/J and A/J, but not BALB/c mice. However, BALB/c progenitors were sensitive to dbcAMP and Forskolin (10(-4) M). In contrast, in precursor assays, PGE(2) (10(-7) - 10(-9) M) blocked responses to IL-5 in bone-marrow from BP-2 and BALB/c mice, either naïve or sensitized and challenged, to a similar extent. PGD(2) (10(-6) M) was ineffective, as was PGE(2) (10(-7) M), if added after 48 h of culture. 4. In conclusion, PGE(2) inhibits the responses of bone-marrow myeloid progenitors to GM-CSF and of eosinophil precursors to IL-5, in naïve or ovalbumin sensitized and challenged mice. These effects are duplicated by cyclic AMP-elevating agents. In the BALB/c strain, the resistance of progenitors, but not precursors, to PGE(2) inhibition, indicates these developmental stages are separate targets for PGE(2) modulation.

Upregulation by glucocorticoids of responses to eosinopoietic cytokines in bone-marrow from normal and allergic mice.

Since the production of eosinopoietic cytokines (GM-CSF, IL-3, IL-5) is inhibited by glucocorticoids, while responsiveness to these cytokines is enhanced in bone-marrow of allergic mice, we studied the ability of glucocorticoids to modulate murine bone-marrow eosinopoiesis. Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized and challenged mice or (c) dexamethasone (1-5 mg kg(-1)) injected mice. Cultures were established with GM-CSF (2 ng ml(-1)) or IL-5 (1 ng ml(-1)), respectively, alone or associated with dexamethasone, hydrocortisone or corticosterone. Total myeloid colony numbers, frequency and size of eosinophil colonies, and numbers of eosinophil-peroxidase-positive cells were determined at day 7. In BALB/c mice, dexamethasone (10(-7) M) increased GM-CSF-stimulated myeloid colony formation (P = 0.01), as well as the frequency (P=0.01) and size (P<0.01) of eosinophil colonies. Dexamethasone (10(-7) M) alone had no effect. Dexamethasone (10(-7)-10(-10) M) increased (P<0.002) eosinophil precursor responses to IL-5. Potentiation by dexamethasone was still detectable: (a) on low density, immature, nonadherent BALB/c bone-marrow cells, (b) on bone-marrow from other strains, and (c) on cells from allergic mice. Hydrocortisone and corticosterone had similar effects. Dexamethasone administered in vivo, 24 h before bone-marrow harvest, increased subsequent progenitor responses to GM-CSF (P = 0.001) and precursor responses to IL-5 (P<0.001). These effects were blocked by RU 486 (20 mg kg(-1), orally, 2 h before dexamethasone, or added in vitro at 10 microM, P<0.001). Glucocorticoids, acting in vivo or in vitro, through glucocorticoid receptors, enhance bone-marrow eosinopoiesis in naïve and allergic mice.

Rapid increase in bone-marrow eosinophil production and responses to eosinopoietic interleukins triggered by intranasal allergen challenge.

To define the effects of immunization and exposure to allergen on the eosinophil lineage, we studied blood and bone-marrow eosinophil numbers, serum interleukin (IL)-5 levels, and eosinophil progenitor and precursor responses to IL-3 and IL-5 in ovalbumin-immunized BALB/c mice after intranasal challenge. Increased blood eosinophilia was found in immune relative to nonimmune mice, but the differences between challenged and unchallenged immune animals were not significant. In contrast, significantly increased circulating levels of IL-5 and numbers of bone-marrow eosinophils were found in sensitized animals exposed to allergen, relative to unchallenged, sensitized controls. An allergen-induced increase in IL-3-sensitive progenitors yielding eosinophil-bearing colonies was also found at 2 h after challenge. Furthermore, an eosinophil differentiation assay showed a marked increase in the magnitude of the responses to IL-5 and IL-3 over a 7-day period in bone-marrow cells of sensitized animals, which was detectable at 24 h after allergen challenge, but not at 2 h and not in unchallenged controls. Modulation of the responses of bone-marrow cells to IL-5 is induced by a circulating factor present in challenged immune animals, as shown by in vivo plasma transfer, but is at best only partly blocked by in vivo treatment with the anti-IL-5 antibody TRFK-5. These data indicate that allergen challenge in the airways leads to rapid long-term modifications in bone-marrow eosinophil progenitors and precursors, and that increased responses to eosinopoietins in bone marrow depend on the release, between 2 h and 24 h after challenge, of a circulating factor distinct from IL-5.