Allergens induce enhanced bronchoconstriction and leukotriene production in C5 deficient mice

BACKGROUND: Previous genetic analysis has shown that a deletion in the complement component 5 gene-coding region renders mice more susceptible to allergen-induced airway hyperresponsiveness (AHR) due to reduced IL-12 production. We investigated the role of complement in a murine model of asthma-like pulmonary inflammation. METHODS: In order to evaluate the role of complement B10 mice either sufficient or deficient in C5 were studied. Both groups of mice immunized and challenged with a house dust extract (HDE) containing high levels of cockroach allergens. Airways hyper-reactivity was determined with whole-body plesthysmography. Bronchoalveolar lavage (BAL) was performed to determine pulmonary cellular recruitment and measure inflammatory mediators. Lung homogenates were assayed for mediators and plasma levels of IgE determined. Pulmonary histology was also evaluated. RESULTS: C5-deficient mice showed enhanced AHR to methylcholine challenge, 474% and 91% increase above baseline Penh in C5-deficient and C5-sufficient mice respectively, p < 0.001. IL-12 levels in the lung homogenate (LH) were only slightly reduced and BAL IL-12 was comparable in C5-sufficient and C5-deficient mice. However, C5-deficient mice had significantly higher cysteinyl-leukotriene levels in the BAL fluid, 1913 +/- 246 pg/ml in C5d and 756 +/- 232 pg/ml in C5-sufficient, p = 0.003. CONCLUSION: These data demonstrate that C5-deficient mice show enhanced AHR due to increased production of cysteinyl-leukotrienes

Human blood basophils produce interleukin-13 in response to IgE- receptor-dependent and -independent activation

Interleukin-13 (IL-13) is a recently discovered immunoregulatory cytokine. The cellular sources of IL-13 and the regulation of its expression are largely unknown. Here we show that human basophils produce IL-13 in response to IgE-receptor (IgER) crosslinking, IL-3, IL-3 plus C5a, but not C5a alone. Human basophils express IL-13 in a restricted manner since, apart from IL-4, no other cytokines encoded on the cytokine gene cluster (IL-3, IL-5, and granulocyte macrophage-colony-stimulating factor [GM-CSF]), are induced. Highest levels of IL-13 are formed after IgE-independent activation leading to a prolonged secretion of IL-13. The response to IgER-cross-linking is more transient preferentially inducing IL-4, IL-3 is a unique cytokine regulating IL-13 production by human basophils: Among a large number of cytokines tested, only IL-3 is capable of directly inducing IL-13 expression. Furthermore, although some IL-13 is produced in response to C5a in the presence of IL-5, GM-CSF, IGF-1 or IL-1 beta, IL-3 is by far the most effective. IL-13 production was blocked by actinomycin D and cycloheximide and conditions leading to IL-13 release also lead to the induction of IL-13 mRNA. This study supports an important immunoregulatory role of human blood basophils, owing to their capacity to simultaneously express IL-13 and IL-4 in a restricted manner.</jats:p

IgE-independent interleukin-4 expression and induction of a late phase of leukotriene C4 formation in human blood basophils

T-helper cells can differentiate into at least two subtypes secreting distinct profiles of cytokines, Th1 and Th2, regulating immunoprotection and different immunopathologies. Interleukin-4 (IL-4) is both the product and the inducer of Th2 cells, raising the question whether IL-4 can be produced in response to antigen-independent stimuli. Here we show that human basophils produce IL-4 on stimulation with IL-3 and C5a or C5adesarg in similar amounts as induced by IgE- receptor-cross-linking. C5a-induced IL-4 production requires the presence of IL-3, with little effect of the sequence of stimuli addition. No “Th1-cytokines” (interferon-gamma and IL-2) and even no “Th2-cytokines” (IL-3, IL-5, IL-10, and granulocyte-macrophage colony- stimulating factor) are produced by basophils in response to either IgE- dependent or IgE-independent activation. The generation of leukotriene C4 (LTC4) is regulated in a similar manner. However, C5a induces a rapid, transient burst of leukotriene formation only if added after IL- 3. Interestingly, upon prolonged culture, a late phase of continuous LTC4 production is observed, which also requires two signals (IL-3 and C5a), but rather depends on their continuous presence than on their sequence of action. These data describe an antigen-independent pathway of very restricted IL-4 expression. Thus, basophils must be considered as central immunoregulatory cells of the innate immune system. Furthermore, the results show that LTC4 can also be generated more continuously for many hours, a phenomenon that may be of particular importance in chornic allergic inflammation, such as asthma.</jats:p

Basophil priming by neurotrophic factors. Activation through the trk receptor

There is increasing evidence that nerve growth factor (NGF) acts on cells of the immune system, apart from its neurotrophic effects. In human basophils, NGF potentiates mediator release and primes the cells to produce leukotriene C4 in response to C5a. It is, however, unknown whether other homologous neurotrophins also act outside the nervous system, and whether activation of basophils by NGF requires interaction with trk tyrosine kinase receptors, the low affinity NGF receptor (LNGFR), or both. A triple mutant NGF designed to interrupt binding to the LNGFR was found to activate basophils with equal efficacy as wild-type NGF, demonstrating that the LNGFR is not necessary. Despite a 10 times lower potency of mutant NGF, no LNGFR expression was detected by FACS analysis. Brain-derived neurotrophic factor, which interacts with trkB, was inactive at concentrations up to 1000 ng/ml (> 30,000-fold lower potency than NGF), while neurotrophin-3, which is thought to interact with trkC, trkB, and more weakly with trk, induced a threshold effect at 300 ng/ml (approximately 10,000-fold lower potency), demonstrating that 1) the LNGFR cannot deliver a direct signal; and 2) basophils do not express functional trkB and trkC receptors. In agreement with the functional data, basophils (in contrast to other granulocyte types) expressed mRNA for trk, but not trkB or trkC, and no or minimal mRNA for LNGFR. These data demonstrate that human blood basophils express functional trk receptors that do not require the participation of LNGFR, and that, among the neurotrophin family, NGF is unique in priming basophils

Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3.

Abstract It has been shown that CC chemokines activate basophil and eosinophil leukocytes with different selectivities and patterns of activity. The most effective are monocyte chemotactic protein-1 (MCP-1), a potent stimulus of mediator release in basophils without effects on eosinophils, RANTES, a weak stimulus of release and strong chemoattractant for basophils and eosinophils, and MCP-3, which combines the activities of MCP-1 and RANTES. We have now compared MCP-2, which has 62 and 60% of sequence identity with MCP-1 and MCP-3, respectively, with the other CC chemokines. MCP-2 induced mediator release by human basophils with lower efficacy and potency than MCP-1 and MCP-3. It promoted transient changes of cytosolic-free calcium concentration ([Ca2+]i) and chemotactic responses in both basophils and eosinophils, however somewhat less efficiently than MCP-3 and RANTES. Desensitization studies indicate that MCP-2 interacts with receptors recognizing MCP-1 as well as RANTES. These results demonstrate that MCP-2 and MCP-3 exert qualitatively similar biologic activities on basophils and eosinophils. In basophils that had not been treated with IL-3, MCP-2 induced minimal exocytosis only, but desensitized the cells toward MCP-1 and MCP-3, suggesting that MCP-2 may act as a functional inhibitor of CC chemokine actions. The results of this study further indicate that MCP analogues display partially distinct, partially overlapping bioactivities toward eosinophils and basophils, and may thus regulate inflammatory processes involving these effector cell types.</jats:p

Basophil priming by neurotrophic factors. Activation through the trk receptor.

Abstract There is increasing evidence that nerve growth factor (NGF) acts on cells of the immune system, apart from its neurotrophic effects. In human basophils, NGF potentiates mediator release and primes the cells to produce leukotriene C4 in response to C5a. It is, however, unknown whether other homologous neurotrophins also act outside the nervous system, and whether activation of basophils by NGF requires interaction with trk tyrosine kinase receptors, the low affinity NGF receptor (LNGFR), or both. A triple mutant NGF designed to interrupt binding to the LNGFR was found to activate basophils with equal efficacy as wild-type NGF, demonstrating that the LNGFR is not necessary. Despite a 10 times lower potency of mutant NGF, no LNGFR expression was detected by FACS analysis. Brain-derived neurotrophic factor, which interacts with trkB, was inactive at concentrations up to 1000 ng/ml (&amp;gt; 30,000-fold lower potency than NGF), while neurotrophin-3, which is thought to interact with trkC, trkB, and more weakly with trk, induced a threshold effect at 300 ng/ml (approximately 10,000-fold lower potency), demonstrating that 1) the LNGFR cannot deliver a direct signal; and 2) basophils do not express functional trkB and trkC receptors. In agreement with the functional data, basophils (in contrast to other granulocyte types) expressed mRNA for trk, but not trkB or trkC, and no or minimal mRNA for LNGFR. These data demonstrate that human blood basophils express functional trk receptors that do not require the participation of LNGFR, and that, among the neurotrophin family, NGF is unique in priming basophils.</jats:p

Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4:detection by flow cytometry

IgE-stimulated cultured basophils from atopic subjects are capable of secreting interleukin-4 (IL-4). We describe a flow-cytometric technique which identified intracellular IL-4 in unstimulated basophils unseparated from peripheral blood mononuclear cells (PBMC) in both atopic (AT) and nonatopic (NC) volunteers