Dolores M. Conroy

Histamine induces cytoskeletal changes in human eosinophils via the H4 receptor

Histamine (0.004–2 μM) induced a concentration‐dependent shape change of human eosinophils, but not of neutrophils or basophils, detected as an increase in forward scatter (FSC) in the gated autofluorescence/forward scatter (GAFS) assay. The histamine‐induced eosinophil shape change was completely abolished by thioperamide (10 μM), an H3/H4 receptor antagonist, but was not inhibited by pyrilamine or cimetidine (10 μM), H1 and H2 receptor antagonists, respectively. The H4 receptor agonists, clobenpropit and clozapine (0.004–2 μM), which are also H3 receptor antagonists, both induced eosinophil shape change, which was inhibited by thioperamide (10 μM). The H3/H4 receptor agonists, imetit, R‐α‐methyl histamine and N‐α‐methyl histamine (0.004–2 μM) also induced eosinophil shape change. Histamine induced actin polymerisation (0.015–10 μM), intracellular calcium mobilisation (10–100 μM) and a significant upregulation of expression of the cell adhesion molecule CD11b (0.004–10 μM) in eosinophils, all of which were inhibited by thioperamide (10–100 μM). In addition, the H4 receptor agonist/H3 receptor antagonist clozapine (20 μM) stimulated a rise in intracellular calcium in eosinophils. Activation of H4 receptors by histamine (1 μM) primed eosinophils for increased chemotactic responses to eotaxin, but histamine (0.1–10 μM) did not directly induce chemotaxis of eosinophils. Pertussis toxin (1 μg ml−1) inhibited shape change and actin polymerisation responses induced by histamine showing that these effects are mediated by coupling to a Gαi/o G‐protein. This study demonstrates that human eosinophils express functional H4 receptors and may provide a novel target for allergic disease therapy.

Eotaxin Is Specifically Cleaved by Hookworm Metalloproteases Preventing Its Action In Vitro and In Vivo

Eotaxin is a potent eosinophil chemoattractant that acts selectively through CCR3, which is expressed on eosinophils, basophils, mast cells, and Th2-type T cells. This arm of the immune system is believed to have evolved to control helminthic parasites. We hypothesized that helminths may employ mechanisms to inhibit eosinophil recruitment, to prolong worm survival in the host. We observed that the excretory/secretory products of the hookworm Necator americanus inhibited eosinophil recruitment in vivo in response to eotaxin, but not leukotriene B4, a phenomenon that could be prevented by the addition of protease inhibitors. Using Western blotting, N. americanus supernatant was shown to cause rapid proteolysis of eotaxin, but not IL-8 or eotaxin-2. N. americanus homogenate was fractionated by gel filtration chromatography, and a FACS-based bioassay measured the ability of each fraction to inhibit the activity of a variety of chemokines. This resulted in two peaks of eotaxin-degrading activity, corresponding to ∼15 and 50 kDa molecular mass. This activity was specific for eotaxin, as responses to other agonists tested were unaffected. Proteolysis of eotaxin was prevented by EDTA and phenanthroline, indicating that metalloprotease activity was involved. Production of enzymes inactivating eotaxin may be a strategy employed by helminths to prevent recruitment and activation of eosinophils at the site of infection. As such this represents a novel mechanism of regulation of chemokine function in vivo. The existence of CCR3 ligands other than eotaxin (e.g., eotaxin-2) may reflect the evolution of host counter measures to parasite defense systems.

Eotaxin and the attraction of eosinophils to the asthmatic lung

Eosinophilic leukocytes accumulate in high numbers in the lungs of asthmatic patients, and are believed to be important in the pathogenisis of asthma. A potent eosinophil chemoattractant is produced in the asthmatic lung. This small protein, the chemokine eotaxin, is synthesized by a number of different cell types, and is stimulated by interleukin-4 and interleukin-13, which are produced by T-helper (Th)2 lymphocytes. Low molecular weight compounds have been developed that can block the eotaxin receptor C-C chemokine receptor (CCR)3, and prevent stimulation by eotaxin. This provides the potential for orally available drugs that can prevent eosinophil recruitment into the lung and the associated damage and dysfunction.

Chemotactic action of prostaglandin E2 on mouse mast cells acting via the PGE2 receptor 3

Mast cells are long-lived cells that are principally recognized for their effector function in helminth infections and allergic reactions. These cells are derived from pluripotential hematopoietic stem cells in the bone marrow that give rise to committed mast cell progenitors in the blood and are recruited to tissues, where they mature. Little is known about the chemotactic signals responsible for recruitment of progenitors and localization of mature mast cells. A mouse model was set up to identify possible mast cell progenitor chemoattractants produced during repeated allergen challenge in vivo. After the final challenge, the nasal mucosa was removed to produce conditioned medium, which was tested in chemotaxis assays against 2-wk murine bone marrow-derived c-kit+ mast cells (BMMC). A single peak of chemotactic activity was seen on reverse-phase HPLC with a retention time and electrospray mass spectrum consistent with prostaglandin E2 (PGE2). This lipid was found to be a highly potent chemoattractant for immature (2-wk) and also mature (10-wk) BMMC in vitro. Fluorescently labeled 2-wk c-kit+ BMMC, when injected intravenously, accumulated in response to intradermally injected PGE2. Analysis using TaqMan showed mRNA expression of the PGE2 receptors 3 (EP3) and 4 (EP4) on 2- and 10-wk BMMC. Chemotaxis induced by PGE2 was mimicked by EP3 agonists, blocked by an EP3 receptor antagonist, and partially inhibited by a MAPKK inhibitor. These results show an unexpected function for PGE2 in the chemotaxis of mast cells.

CCR4 blockade does not inhibit allergic airways inflammation

The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen‐challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage‐derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen‐specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.

Identification of Selective Basophil Chemoattractants in Human Nasal Polyps as Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca2+ flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.

Chronic ‘Immunological’ Rhinosinusitis: General Aspects, Cytokines, Chemokines and Possible Therapeutic Consequences

Chronic rhinosinusitis is an inflammatory condition of the nose and the paranasal sinuses characterized by an infiltration of neutrophils, eosinophils in addition to lymphocytes (T), mast cells and macrophages. An international clinical definition of chronic rhinosinusitis exists, but not on an immunological basis. Chronic rhinosinusitis has different etiologies with consequently different immunological inflammatory mediator results. These inflammation mediators are responsible for propagating the inflammation. The complex interactions among these cells are mediated by a group of secreted low-molecular-weight proteins that are collectively designated as cytokines to denote their role in cell-to-cell communication. Cytokines assist in regulating the development of immune effector cells, act locally and some cytokines possess direct effector functions of their own. Higher levels of IL-1β, -3, -4, -5, -6, GM-CSF, IFN-γ, TNF-α and TGF-β have been reported in chronic rhinosinusitis compared to control tissues, but in the literature no concordant results have been found, possibly due to inhomogeneous groups. The attraction of leukocytes to tissues is essentially guided by chemokines (chemotactic cytokines). IL-8, MCP-3 and MCP-4, eotaxins 1 and 2 are increased, but as for the other cytokines no concordant results are diposable today.

Eotaxin in Disease

Asthma is a common disease affecting approx 10% of the population in developed countries, and the prevalence and mortality are rising. The disease has characteristic symptoms, namely intermittent airway obstruction, airway hyperresponsiveness, and increased numbers of activated inflammatory cells and airway structural changes as a result of chronic airway inflammation. The primary underlying abnormality in bronchial asthma is thought to be the unique form of airway inflammation, including particularly eosinophils and mast cells, that gives rise to reversible airway obstruction and hyperresponsiveness.