Peter J. Jose

Neutrophil chemoattractants generated in two phases during reperfusion of ischemic myocardium in the rabbit. Evidence for a role for C5a and interleukin-8.

The neutrophil chemoattractants generated in a model of myocardial infarction in the anesthetized rabbit were investigated. Coronary artery occlusion was followed by reperfusion for periods from 5 min to 4.5 h. Extracts of myocardial tissue in normal and post-ischemic zones were tested for C5a and interleukin-8 (IL-8) using specific radioimmunoassays. In the post-ischemic zone, immunoreactive C5a was detected within 5 min and rose progressively to reach a plateau at 3-4.5 h. In contrast, immunoreactive IL-8 concentrations rose after a delay and were highest at the last time point tested, 4.5 h. Myeloperoxidase activity levels, an index of neutrophil accumulation, rose progressively as the concentrations of chemoattractants increased. Using cation exchange and reversed phase HPLC, immunoreactive C5a and IL-8 co-eluted with authentic standards. Fractions taken at the C5a and IL-8 peaks from reversed phase HPLC exhibited neutrophil aggregating activity which was neutralized by the respective antibody used in the radioimmunoassays. Depletion of circulating neutrophils virtually abolished immunoreactive IL-8 in the post-ischemic myocardial tissue. These observations suggest a sequential release of chemoattractants: the first, C5a is generated in interstitial fluid, followed by IL-8 generated by infiltrating neutrophils. Thus, over the time period studied, IL-8 generation would be expected to be indirectly dependent on C5a production.

Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors

Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit+ population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B4. Other activation products were inactive. LTB4 was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB4 receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB4. Furthermore, LTB4 was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB4 was a potent chemoattractant for human cord blood–derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB4 in regulating tissue mast cell numbers.

Cutting Edge: Lipoxin (LX) A4 and Aspirin-Triggered 15-Epi-LXA4 Block Allergen-Induced Eosinophil Trafficking

Tissue eosinophilia prevention represents one of the primary targets to new anti-allergic therapies. As lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous “stop signals” produced in distinct pathologies including some eosinophil-related pulmonary disorders, we evaluated the impact of in situ LXA4/ATL metabolically stable analogues on allergen-induced eosinophilic pleurisy in sensitized rats. LXA4/ATL analogues dramatically blocked allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, inhibiting the earlier edema and neutrophilia associated with allergic reaction. The mechanisms underlying this LXA4/ATL-driven allergic eosinophilia blockade was independent of mast cell degranulation and involved LXA4/ATL inhibition of both IL-5 and eotaxin generation, as well as platelet activating factor action. These findings reveal LXA4/ATL as a novel class of endogenous anti-allergic mediators, capable of preventing local eosinophilia.

Tear and mucus eotaxin-1 and eotaxin-2 in allergic keratoconjunctivitis

OBJECTIVE Eotaxin-1 and eotaxin-2 are potent eosinophil chemotactic and activating peptides that may be implicated in the pathogenesis of the chronic allergic eye diseases vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). The purpose of this study was to measure these chemokines in tear and mucus samples of active-disease patients and in vitro cultured conjunctival epithelial cells and fibroblasts. DESIGN Comparative, observational case series and in vitro study. PARTICIPANTS Sixteen patients with clinically active and untreated VKC or AKC, six age-matched control patients, and five nonactive seasonal allergic conjunctivitis patients. METHODS Tears were collected from the active VKC and AKC patients, and from the normal patients. Mucus was collected from six of these VKC patients. Tears were also collected from an additional five allergic patients after obtaining a positive reaction to conjunctival allergen challenge. Conjunctival epithelial cell and conjunctival fibroblast cultures were exposed to interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-alpha), or to combinations of these cytokines. MAIN OUTCOME MEASURES Levels of eotaxin-1 and eotaxin-2 in tears, mucus, and culture medium. RESULTS High levels of eotaxin-1 and eotaxin-2 were found in mucus of VKC patients, whereas only eotaxin-2 was found to have increased significantly in tears of VKC and AKC patients compared with those of normal patients. Mucus contained higher levels of chemokines than did tears. Both tear eotaxin-1 and eotaxin-2 were correlated significantly with the percent of eosinophils in tear fluid. Eotaxin-1 also was correlated significantly with the sum clinical score and corneal involvement in VKC patients. Conjunctival epithelial cells in culture did not produce eotaxin-1 or eotaxin-2, either at baseline or after cytokine exposure. Conjunctival fibroblasts produced eotaxin-1 only after exposure to IL-4, TNF-alpha, and the combination of IL-4 plus TNF-alpha or IL-13 plus TNF-alpha. CONCLUSIONS Eotaxin-1 and eotaxin-2 are implicated in eosinophil recruitment and in the pathogenesis of VKC and AKC. Cytokine-stimulated conjunctival fibroblasts may be one source of eotaxin-1 in severely allergic tissues.

Neuronal eotaxin and the effects of ccr3 antagonist on airway hyperreactivity and M2 receptor dysfunction

Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.

Induction of eotaxin expression and release from human airway smooth muscle cells by IL‐1β and TNFα: effects of IL‐10 and corticosteroids

Eotaxin is a novel C‐C chemokine with selective chemoattractant activity for eosinophils. We determined whether eotaxin could be produced by human airway smooth muscle (HASM) cells in culture and examined its regulation by interleukin‐10 (IL‐10) and the corticosteroid, dexamethasone. Stimulation of the cells with interleukin‐1β (IL‐1β) or tumour necrosis factor (TNFα) each at 10 ng ml−1 induced the release of eotaxin protein with maximal accumulation by 24 h. Interferon‐γ (IFNγ) alone at 10 ng ml−1 had no effect and there was no synergy between these cytokines on the release of eotaxin. Reverse phase high performance liquid chromatographic (HPLC) analysis of supernatents from cells treated with TNFα (10 ng ml−1 for 96 h showed immunoreactivity to eotaxin which eluted with the expected retention time of 34.5–35 min. Both IL‐1β and TNFα‐induced release of eotaxin was not inhibited by dexamethasone (1 μM), however IL‐10 (10 ng ml−1) had a significant inhibitory effect. Dexamethasone and IL‐10 did not inhibit the induction of eotaxin mRNA induced by IL‐1β or TNFα. Thus, human airway smooth muscle cells can release eotaxin and could be an important source of chemokine production during airway inflammatory events.

Angiotensin II Induces Neutrophil Accumulation In Vivo Through Generation and Release of CXC Chemokines

Background—Angiotensin II (Ang II) is implicated in the development of cardiac ischemic disorders in which prominent neutrophil accumulation occurs. Ang II can be generated intravascularly by the renin-angiotensin system or extravascularly by mast cell chymase. In this study, we characterized the ability of Ang II to induce neutrophil accumulation. Methods and Results—Intraperitoneal administration of Ang II (1 nmol/L) induced significant neutrophil recruitment within 4 hours (13.3±2.3×106 neutrophils per rat versus 0.7±0.5×106 in control animals), which disappeared by 24 hours. Maximal levels of CXC chemokines were detected 1 hour after Ang II injection (577±224 pmol/L cytokine-inducible neutrophil chemoattractant [CINC]/keratinocyte-derived chemokine [KC] versus 5±3, and 281±120 pmol/L macrophage inflammatory protein [MIP-2] versus 14±6). Intravital microscopy within the rat mesenteric microcirculation showed that the short-term (30 to 60 minutes) leukocyte–endothelial cell interactions induced by Ang II were attenuated by an anti-rat CINC/KC antibody and nearly abolished by the CXCR2 antagonist SB-517785-M. In human umbilical vein endothelial cells (HUVECs) or human pulmonary artery media in culture, Ang II induced interleukin (IL)-8 mRNA expression at 1, 4, and 24 hours and the release of IL-8 at 4 hours through interaction with Ang II type 1 receptors. When HUVECs were pretreated with IL-1 for 24 hours to promote IL-8 storage in Weibel-Palade bodies, the Ang II–induced IL-8 release was more rapid and of greater magnitude. Conclusions—Ang II provokes rapid neutrophil recruitment, mediated through the release of CXC chemokines such as CINC/KC and MIP-2 in rats and IL-8 in humans, and may contribute to the infiltration of neutrophils observed in acute myocardial infarction.

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.

Menopause and Ovariectomy Cause a Low Grade of Systemic Inflammation that May Be Prevented by Chronic Treatment with Low Doses of Estrogen or Losartan

The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-β-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. Increased circulating levels of IL-8, MCP-1, RANTES, and MIP-1α and monocyte CD11b expression were also encountered in postmenopausal vs premenopausal subjects. This translational data led us to investigate the mechanisms in Sprague-Dawley rats. Using intravital microscopy, we imaged mesenteric arterioles and found significant increases in arteriolar leukocyte adhesion, cell adhesion molecule expression, and plasma levels of cytokine-induced neutrophil chemoattractant (CINC/KC), MCP-1, and MIP-1α in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-β-estradiol, losartan, both, or benazepril inhibited ovariectomy-induced arteriolar mononuclear leukocyte adhesion by 77%, 58%, 92%, and 65% respectively, partly by inhibition of cell adhesion molecule up-regulation and the increase in circulating chemokines. These results demonstrate that menopause and ovariectomy generate a low grade of systemic inflammation. Therefore, administration of low doses of estrogens or inhibition of the renin-angiotensin system, at early stages of estrogen deficiency, might prevent the systemic inflammation associated with menopause and decrease the risk of suffering further cardiovascular diseases.

Eotaxin-2 generation is differentially regulated by lipopolysaccharide and IL-4 in monocytes and macrophages

The eotaxins are a family of CC chemokines that coordinate the recruitment of inflammatory cells, in particular eosinophils, to sites of allergic inflammation. The cDNA for eotaxin-2 (CC chemokine ligand 24) was originally isolated from an activated monocyte library. In this study, we show for the first time that peripheral blood monocytes generate bioactive eotaxin-2 protein constitutively. Eotaxin-2 production was significantly up-regulated when monocytes were stimulated with the proinflammatory cytokine IL-1β and the microbial stimuli, LPS and zymosan. In contrast, the Th2 cytokines, IL-4 and IL-13, and the proinflammatory cytokine, TNF-α, acting alone or in combination, did not enhance the generation of eotaxin-2 by monocytes. Indeed, IL-4 suppressed the generation of eotaxin-2 by LPS-stimulated monocytes. Although other chemokines, including macrophage-inflammatory protein-1α, monocyte chemoattractant protein-1, macrophage-derived chemokine, and IL-8 were generated by monocytes, eotaxin-1 (CC chemokine ligand 11) could not be detected in the supernatants of monocytes cultured in the presence or absence of any of the stimuli used in the above experiments. Furthermore, human dermal fibroblasts that produce eotaxin-1 did not generate eotaxin-2 under basal conditions or when stimulated with specific factors, including IL-4, IL-13, TNF-α, and LPS. When monocytes were differentiated into macrophages, their constitutive generation of eotaxin-2 was suppressed. Moreover, IL-4, but not LPS, up-regulated the production of eotaxin-2 by macrophages. Taken as a whole, these results support a role for macrophage-derived eotaxin-2 in adaptive immunity, with a Th2 bias. In contrast, a role for monocyte-derived eotaxin-2 is implicated in innate immunity.