Eotaxin protein and gene expression in guinea-pig lungs: constitutive expression and upregulation after allergen challenge.

Eotaxin is an eosinophil-specific chemoattractant originally identified in bronchoalveolar lavage fluid after allergen challenge of sensitized guinea-pigs. We have determined and quantified for the first time the cellular sources of guinea-pig lung eotaxin and localized gene expression in structural cells of large and small airways and in alveolar macrophages. We used anti-guinea-pig eotaxin monoclonal and polyclonal antibodies and a complementary ribonucleic acid (cRNA) probe to detect eotaxin protein and cytoplasmic messenger ribonucleic acid (mRNA) transcripts by the techniques of immunohistochemistry and in situ hybridization in: 1) naive; 2) ovalbumin-sensitized/ saline-exposed; and 3) ovalbumin-sensitized/ovalbumin-challenged animals (n = 5 for each group). Compared with the naive animals, there was a fivefold increase of eotaxin protein and a 25 fold upregulation of eotaxin gene expression in the airway epithelium 3 h after ovalbumin challenge of sensitized animals (p < 0.001). The average percentages of alveolar macrophages staining for eotaxin protein and mRNA in the naive animals were approximately 30 and 10% respectively: both increased significantly in the sensitized/ovalbumin-challenged animals to 78 and 57%, respectively (p < 0.0001). Compared with the naive animals, the procedure of sensitization and saline exposure significantly increased eotaxin gene expression in both bronchial epithelium and alveolar macrophages (p < 0.01): the upregulation at these two sites showed a strong positive association (rcorr = 0.95; p < 0.0001). The results indicate that there are multiple cellular sources of guinea-pig lung-derived eotaxin, including bronchial and bronchiolar epithelial cells, airway smooth muscle, bronchial vascular endothelium, and chondrocytes and alveolar macrophages, and that there are relatively rapid and marked increases of eotaxin protein and gene expression in airway epithelium and alveolar macrophages following allergen challenge.

Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo.

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.

Animal models of asthma: role of chemokines.

In studies of disease processes, increasing knowledge leads to an increased awareness of the complexity of the underlying mechanisms. The intense research activity in the chemokine field has made this acutely manifest. Numerous chemokines have been discovered through the use of (1) bioassay of in vitro cell culture supernatants and in vivo exudates from animal models of inflammation and (2) molecular biology techniques. Any one chemokine can often be produced by a number of different cell types and exert its effects on different target cells. This has been interpreted by some as implying a high degree of redundancy. Although this is understandable, in disease processes parallel and sequential mechanisms are possible, and potentially important therapeutic targets have emerged. There is compelling evidence from animal and clinical studies that eosinophils are important effector cells in asthma, but this relationship is as yet unproven in the human disease. Two possible targets to prevent eosinophil recruitment to the lung are IL-5 and its receptor, which are important in several aspects of eosinophil biology, and eotaxin and its receptor, CCR3. The eotaxin receptor is particularly attractive as a target as it is expressed in high numbers on eosinophils, but not other leukocytes, and appears to be the major detector of the eosinophil for eotaxin and other chemokines such as MCP-4. Eotaxin and CCR3 knockout mice are being developed, and animal models will continue to be invaluable when antagonists are available. In the shape of receptor antagonists, the chemokine field may yet provide the final proof of concept for the long-established eosinophil theory of asthma in humans.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation.

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralleled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer and attractive target for therapeutic intervention.

Human RANTES acts as a receptor antagonist for guinea pig eotaxin in vitro and in vivo.

The guinea pig C-C chemokine, eotaxin, is a potent and selective eosinophil chemoattractant in guinea pig airways and skin in vivo, and stimulates both guinea pig and human eosinophils in vitro. The human C-C chemokine RANTES (30% homology with guinea pig eotaxin) stimulates human eosinophils in vitro, but does not stimulate guinea pig eosinophils, even though these cells bind 125I-RANTES. Similar concentrations of eotaxin and unlabeled RANTES competitively inhibit the binding of 125I-RANTES to guinea pig eosinophils, suggesting that eotaxin and RANTES share a common binding site on these cells. In the present study, we investigated the possibility that human RANTES, binding to a putative eotaxin receptor on guinea pig eosinophils, might block functional responses to eotaxin. When fura-2-loaded cells were first exposed to RANTES, which failed to elevate the intracellular calcium concentration, the response to a subsequent challenge with eotaxin was inhibited in a dose-dependent manner. Inhibition was also demonstrated when the two chemokines were added simultaneously. Another human C-C chemokine, MCP-3 (52% homology with guinea pig eotaxin), had similar inhibitory effects on the eotaxin-induced activation of guinea pig eosinophils in vitro. RANTES inhibited (111)In-eosinophil accumulation in response to intradermal eotaxin in vivo. In contrast, RANTES had no significant effect on responses to leukotriene B4 in vitro or in vivo. Thus, these experiments in the guinea pig demonstrate that human RANTES is the first prototypic antagonist of an eotaxin receptor.

Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.

Experiments 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 B4. 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.

Eosinophil chemoattractants generated in vivo.

The eosinophil is the predominant inflammatory cell which accumulates in the asthmatic lung. There is considerable circumstantial evidence linking these cells to lung dysfunction, but the precise cause and effect relationship is controversial. The defensive role of the eosinophil appears to be concerned largely with eliminating helminth parasites which do not normally present a constant threat. Thus, unlike the neutrophil whose defensive role against microbes is essential, the eosinophil presents a target for therapeutic intervention which is potentially applicable to long-term treatment. Several approaches to suppressing eosinophil accumulation are possible, based on the multiple steps involved in their appearance and activation in the lung (for review see [1]). One approach is to block the receptor(s) to the important endogenous eosinophil chemoattractants generated in the asthmatic lung, offering the potential for selective leukocyte-type suppression. A first step in this pursuit is the identification of such chemoattractants. This article describes recent attempts in this direction, with the long-term goal of producing chemoattractant receptor antagonists.

Eotaxin: cloning of an eosinophil chemoattractant cytokine and increased mRNA expression in allergen-challenged guinea-pig lungs.

Eotaxin was recently identified as the major eosinophil chemoattractant in bronchoalveolar lavage fluid obtained 3h after allergen challenge of sensitised guinea-pigs. We now report the cDNA cloning of this C-C chemokine. The 777 base-pair clone, pEo3122, consists of a 40 base 5' untranslated region, an open reading frame of 288 bases predicting a 73 amino acid mature protein plus a 23 amino acid signal peptide, and a 3' untranslated region of 449 bases containing a poly A tail. Northern blot analysis showed eotaxin mRNA in the lungs of naive and sensitised guinea-pigs, which was considerably increased after allergen challenge. Eotaxin may be an important mediator of eosinophil accumulation and activation in allergic reactions. As eotaxin stimulates human eosinophils, this chemokine and related molecules may be involved in human diseases such as asthma where eosinophil accumulation is a prominent feature.

Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation.

Eosinophil 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-1 alpha, 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-1 alpha, and MCP-1 were all inactive in inducing 111In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with RANTES 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.

The chemokine, eotaxin, activates guinea-pig eosinophils in vitro and causes their accumulation into the lung in vivo.

Eotaxin is a novel C-C chemokine purified from the bronchoalveolar lavage (BAL) fluid of actively sensitised guinea-pigs after aerosol allergen challenge. In this study we show that eotaxin induced a dose-dependent increase in both intracellular-free calcium concentration ([Ca2+]i) and aggregation of guinea-pig eosinophils in vitro. Intradermally injected eotaxin induced the accumulation of [111In]eosinophils in naive guinea-pig skin in vivo, without oedema-inducing activity; the latter emerging in separate BAL fluid HPLC fractions. Aerosol exposure of naive guinea-pigs to eotaxin in vivo caused a selective increase in eosinophils in BAL fluid. Thus, eotaxin activates guinea-pig eosinophils in vitro and causes a selective eosinophil accumulation in the lung in vivo. Eotaxin and related molecules are potentially important endogenous signalling substances in allergic reactions in vivo.

Validation of a non-invasive technique to assess development of airway hyperreactivity in an animal model of immunologic pulmonary hypersensitivity.

Airway hyperreactivity (AHR) is considered to be a prominent and consistent feature of the asthmatic. Accordingly, in developing animal models of asthma, it is important to have methodologies available for repeated assessment of airway reactivity (AR). We have described a methodology to assess AR in conscious minimally restrained guinea pigs, AR being quantified as the airborne concentration of histamine (mg m-3) necessary to produce a mild airway constriction. The present study sought to validate that methodology by assessing its ability to detect changes in AR associated with immediate-onset pulmonary hypersensitivity responses. Guinea pigs were sensitized by intraperitoneal injection of ovalbumin (OA) and challenged with OA aerosol 3 weeks later. All animals developed severe immediate-onset airway constrictive responses. AR was assessed 1 h later, upon return to normal breathing patterns. Hyperreactivity was apparent from response to 0.50 mg m-3 histamine when compared with 2.10 mg m-3 histamine needed for baseline response. In control, sham sensitized animals AR remained at 2.12 mg m-3 after OA inhalation challenge. The results demonstrate the ability of this methodology to detect airway hyperreactivity to histamine resulting from a pulmonary hypersensitivity response. By requiring neither surgery nor any invasive procedure, the technique is appropriate for serial measurements of AR as is needed in development of an animal model for asthma, a chronic airway disease.

Measurement of specific airway conductance in guinea pigs. A noninvasive method.

A constant volume plethysmographic technique has been developed to measure specific airway conductance (sGaw) in unanesthetized spontaneously breathing guinea pigs. The technique utilizes a specially designed animal restraining device and mask piece. sGaw is measured at end-expiration and does not require knowledge of thoracic gas volume. Control values are within the range reported previously for this species. The method is noninvasive with minimum stress to the animals. Exposure of guinea pigs to an aerosol of cotton dust extract produces similar qualitative changes (a fall) in sGaw to those observed in human volunteers exposed to the same aerosol. The method is proposed as a suitable model for the study of byssinosis (the occupational lung disease associated with chronic exposure to cotton dust). The technique may also be applied to the acute and chronic study of the airway response to other airborne pharmacological and toxicological agents.