SllmmsLryEosinophil accumulation is a prominent feature of allergic inflammatory reactions, such as those occurring in the lung of the allergic asthmatic, but the endogenous chemoattractants involved have not been identified. We have investigated this in an established model of allergic inflammation, using in vivo systems both to generate and assay relevant activity. Bronchoalveolar lavage (BAL) fluid was taken from sensitized guinea pigs at intervals after aerosol challenge with ovalbumin. BAL fluid was injected intradermally in unsensitized assay guinea pigs and the accumulation of intravenously injected 111In-eosinophils was measured. Activity was detected at 30 min after allergen challenge, peaking from 3 to 6 h and declining to low levels by 24 h. 3-h BAL fluid was purified using high performance liquid chromatography techniques in conjunction with the skin assay. Microsequencing revealed a novel protein from the C-C branch of the platelet factor 4 superfamily of chemotactic cytokines. The protein, "eotaxin," exhibits homology of 53% with human MCP-1, 44% with guinea pig MCP-1, 31% with human MIP-loe, and 26% with human RANTES. Laser desorption time of flight mass analysis gave four different signals (8.15, 8.38, 8.81, and 9.03 kD), probably reflecting differential O-glycosylation. Eotaxin was highly potent, inducing substantial 111In-eosinophil accumulation at a 1-2-pmol dose in the skin, but did not induce significant 111In-neutrophil accumulation. Eotaxin was a potent stimulator of both guinea pig and human eosinophils in vitro. Human recombinant RANTES, MIP-lcr, and MCP-1 were all inactive in inducing ~11In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with P.ANTES on guinea pig eosinophils. This is the first description of a potent eosinophil chemoattractant cytokine generated in vivo and suggests the possibility that similar molecules may be important in the human asthmatic lung.

SummaryExperiments were designed to study the effect of systemically administered IL-5 on local eosinophil accumulation induced by the intradermal injection of the chemokine eotaxin in the guinea pig. Intravenous interleukin-5 (IL-5) stimulated a rapid and dramatic increase in the numbers of accumulating eosinophils induced by i.d.-injected eotaxin and, for comparison, leukotriene B 4. The numbers of locally accumulating eosinophils correlated directly with a rapid increase in circulating eosinophils: circulating eosinophil numbers were 13-fold higher 1 h after intravenous IL-5 (18.3 pmol/kg). This increase in circulating cells corresponded with a reduction in the number of displaceable eosinophils recovered after flushing out the femur bone marrow cavity. Intradermal IL-5, at the doses tested, did not induce significant eosinophil accumulation. We propose that these experiments simulate important early features of the tissue response to local allergen exposure in a sensitized individual, with eosinophil chemoattractant chemokines having an important local role in eosinophil recruitment from blood microvessels, and IL-5 facilitating this process by acting remotely as a hormone to stimulate the release into the circulation of a rapidly mobilizable pool of bone marrow eosinophils. This action of IL-5 would be complementary to the other established activities of IL-5 that operate over a longer time course.

We determined the effect of inhaled corticosteroid, budesonide, on the release of the anti-inflammatory cytokine, interleukin-10 (IL-10), and of pro-inflammatory cytokines, macrophage inflammatory protein-1alpha (MIP-1alpha), interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF), from blood monocytes and alveolar macrophages of mild asthmatic subjects in a double-blind, cross-over, placebo-controlled study. Budesonide reduced bronchial hyperresponsiveness and improved baseline FEV1. Alveolar macrophages were obtained by bronchoalveolar lavage performed at the end of each treatment phase. IL-10 from blood monocytes was not altered, but both IL-10 mRNA and protein expression from alveolar macrophages stimulated by lipopolysaccharide and IL-1beta were increased after corticosteroid therapy. By contrast, alveolar macrophages released significantly less MIP-1alpha, IFN-gamma, and GM-CSF after steroid treatment. In comparison to alveolar macrophages from normal nonasthmatic volunteers, those from asthmatic patients released more MIP-1alpha, IFN-gamma, and GM-CSF but lower amounts of IL-10 particularly at baseline and after IL-1beta stimulation. The ability of steroids to inhibit pro-inflammatory cytokines but to enhance the anti-inflammatory cytokine such as IL-10 may contribute to their beneficial actions in asthma. Asthma is characterized by alveolar macrophages exhibiting both an enhanced capacity to release pro-inflammatory cytokines and a reduced capacity to produce IL-10.

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12–24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge.

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.

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.

These data indicate that potent amplification of inflammation induced by TLR agonists, such as LPS, may be achieved by cooperativity between airway smooth muscle and leukocytes involved in immune surveillance or inflammation.

Interleukin (IL)-8 is a C-X-C chemokine that potently chemoattracts and activates neutrophils. We determined whether IL-8 could be produced by human airway smooth muscle cells in culture and examined its regulation. TNF-alpha stimulated IL-8 mRNA expression and protein release in a time- and dose-dependent manner, whereas IFN-gamma alone had no effect. Both cytokines together did not induce greater IL-8 release compared to TNF-alpha alone. IL-1beta was more potent in inducing IL-8 release and, together with TNF-alpha, there was a synergistic augmentation of IL-8 release. IL-8 release induced by TNF-alpha and IFN-gamma was partly inhibited by the Th-2-derived cytokines IL-4, IL-10, and IL-13, as well as by dexamethasone. In addition to its contractile responses, airway smooth muscle cells have synthetic and secretory potential with the release of IL-8 and subsequent recruitment and activation of neutrophils in the airways. Release of IL-8 can be modulated by Th-2-derived cytokines and corticosteroids.