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.

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 β2 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.

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.

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)-1α. PD98059 blocked C5a- but not ionomycin-induced MIP-1α production (59% inhibition at 50 μM concentration). In contrast, SB202190 nearly completely inhibited (99%) C5a-induced MIP-1α 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.

We demonstrate that mitogen-activated protein kinase–activated kinase-2 (MK2) is essential for localized Th2-type inflammation and development of experimental asthma. MK2 deficiency does not affect systemic Th2 immunity, but reduces endothelial permeability, as well as adhesion molecule and chemokine expression. NF-κB regulates transcription of adhesion molecules and chemokines. We show that MK2 and its substrate HSP27 are essential for sustained NF-κB activation. MK2 and HSP27 prevent nuclear retention of p38 by sequestering it in the cytosol. As a result, MK2 precludes excessive phosphorylation of MSK1. By reducing MSK1 activity, MK2 prevents p65 NF-κB hyperphosphorylation and excessive IκBα transcription. IκBα mediates nuclear export of p65. By reducing IκBα level, MK2 prevents premature export of NF-κB from the nucleus. Thus, the MK2–HSP27 pathway regulates the NF-κB transcriptional output by switching the activation pattern from high level, but short lasting, to moderate-level, but long lasting. This pattern of activation is essential for many NF-κB–regulated genes and development of inflammation. Thus, the MK2–HSP27 pathway is an excellent target for therapeutic control of localized inflammatory diseases.

Lyn, an Src-type tyrosine kinase, is associated with the interleukin (IL)-5 receptor in eosinophils. The mechanism of its activation is unknown. Through yeast twohybrid screening we have cloned and characterized a new signaling molecule, Unc119, that associates with IL-5R␣ and Src family tyrosine kinases. Unc119 induces the catalytic activity of these kinases through interaction with Src homology 2 and 3 domains. IL-5 stimulation of eosinophils increases Unc119 association with Lyn and induces its catalytic activity. Lyn is important for eosinophil survival. Eosinophils that are transduced with Unc119 have increased Lyn activity and demonstrate prolonged survival in the absence of IL-5. Inhibition of Unc119 down-regulates eosinophil survival. To our knowledge Unc119 is the first receptor-associated activator of Src family tyrosine kinases.One of the fundamental goals of cell biology is to understand the mechanism of signal generation by receptors. Many receptors rely upon kinases, especially tyrosine kinases, for receptor phosphorylation and activation of signaling cascades. The Src family tyrosine kinases (SrcTKs) 1 frequently serve as the trigger mechanism for cytosolic signals (1). Receptor-associated SrcTKs exist in a non-active conformation and become transiently activated following ligand binding and receptor oligomerization (2). How receptor oligomerization leads to the activation of SrcTKs is unknown.Two intramolecular interactions tightly regulate structural conformation and enzymatic activity of SrcTKs. One is through Src homology 2 (SH2) and the other is through the SH3 domain. All cellular SrcTKs have a C-terminal regulatory tyrosine residue (Tyr 527 for Src). Phosphorylated Tyr 527 forms an intramolecular interaction with the SH2 domain of the kinase (3). This interaction decreases the kinase activity. Dephosphorylation of this tyrosine residue by phosphatases such as CD45 is required but is not sufficient for kinase activation (4). Receptor-bound SrcTKs are frequently found to be dephosphorylated under basal conditions, yet lack appreciable catalytic activity (5). CD45-negative cells have variable effects on SrcTKs. CD45 negatively regulates Lyn by dephosphorylating both its negative and positive regulatory tyrosine residues (6). The activity of Lyn and Hck is increased in macrophages (7) and B cells (8) lacking CD45.Regulation of SrcTK activity through other intramolecular interactions was realized after the crystal structures of Src (9) and Hck (10) were solved. The linker region that is located between the SH2 and the kinase domains binds to the SH3 domain of the kinase rendering a non-active conformation. Furthermore, a short amino acid stretch connecting the SH2 and SH3 domains has recently been identified as an additional negative regulator of Hck and Src activation (11). The SH3 domain of SrcTKs prefers to bind to the RXXPXXP motif (a motif is defined as a short stretch of amino acid residues that binds to a signaling domain) (12, 13). Although the linkers of SrcTK bind to the SH3 domain, ...

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 &gt; 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 &gt; 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.

SummaryMacrophage inflammatory protein-1 (MIP) is a recently cloned cytokine that causes neutrophilic infiltration and induces an inflammatory response. We studied the effect of MIP-lo~ on histamine secretion from basophils and mast cells. Leukocytes from allergic and normal subjects were studied. MIP-loe caused dose-dependent release of histamine from basophils of 14 of 20 allergic donors at concentrations of 10-9-10 -7 M, and the mean release was 13.50 + 2.9% at the highest concentration. In the same experiments, the mean histamine release by anti-immunoglobulin E and monocyte chemotactic and activating factor (MCAF) (10 -7 M) was 32 + 7% and 31 _+ 3%, respectively. The cells from only 2 of 10 normal subjects released histamine in response to MIP-loe. Histamine release by MIP-lo~ was rapid, and almost complete within the first 3 rain. MIP-lce-induced degranulation was a calcium-dependent noncytotoxic process. MIP-lo~ showed chemotactic activity for purified basophils that was comparable to MCAF. Both MIP-lo~ and MCAF at 10-7 M concentration elicited a chemotactic response that was 40% of the maximal response to CSa (1/zg/ml). Murine MIP-lo~ induced histamine release from mouse peritoneal mast cells in a dose-dependent manner. Thus, we have established that MIP-lo~ is a novel activator of basophils and mast cells.