Increase effect of transforming growth factor on eotaxin production by normal cultured dermal fibroblasts stimulated with interleukin-4: inhibitory effect of suplatast tosilate on eotaxin production.

Eotaxin plays a central role in the development of allergic disease, including atopic dermatitis, asthma, and nasal allergy. Interleukin (IL)-4 induces eotaxin production in normal human dermal fibroblasts. On the other hands, Transforming growth factor-beta (TGF-beta), a multifunctional regulatory cytokine, affects many biological functions, including fibroblast growth and differentiation and Th2 cytokine regulation. In this study, we investigated the effect of TGF-beta on IL-4-induced eotaxin production by normal human fibroblasts, as well as the effect of suplatast tosilate, an antiallergic drug that selectively inhibits Th2 cytokine production. Dermal fibroblast treatment with IL-4 and TGF-beta for 24 h increased eotaxin production and expression of eotaxin mRNA, as measured by enzyme-linked immunosorbent assay (ELISA) and reverse-transcriptase polymerase chain reaction (RT-PCR), respectively. TGF-beta synergistically up-regulated eotaxin production and eotaxin mRNA expression when stimulated with IL-4. Suplatast tosilate dose-dependently inhibited eotaxin production induced by IL-4 or IL-4 plus TGF-beta. These results suggest that TGF-beta may regulate skin allergic inflammation by up-regulating eotaxin production in dermal fibroblasts. Suplatast tosilate might suppress this inflammation by inhibiting eotaxin production.

Treatment with a novel chemokine-binding protein or eosinophil lineage-ablation protects mice from experimental colitis.

Eosinophils are multifunctional leukocytes implicated in numerous inflammatory diseases. The present study was conducted to clarify the precise role of eosinophils in the development of colitis by using eosinophil-depleted mice and a novel chemokine-binding protein that neutralizes CCL11 action. Colitis was induced by administration of dextran sodium sulfate (DSS) to wild-type and eosinophil-deficient DeltadblGATA-1 mice. Accumulation of eosinophils in the gut of mice given DSS paralleled worsening of clinical score and weight loss. In response to DSS, DeltadblGATA-1 mice showed virtual absence of eosinophil recruitment, amelioration of clinical score, weight loss, and tissue destruction, and no lethality. There was a decrease in CXCL1 and CCL3 production and decreased neutrophil influx in the intestine of DeltadblGATA-1 mice. Transfer of bone marrow cells from wild-type mice reconstituted disease manifestation in DSS-treated DeltadblGATA-1 mice, and levels of CCL11 were increased after DSS treatment and localized to inflammatory cells. Treatment with the chemokine-binding protein evasin-4 at a dose that prevented the function of CCL11 greatly ameliorated clinical score, weight loss, overall tissue destruction, and death rates. In conclusion, the influx of eosinophils is critical for the induction of colitis by DSS. Treatment with a novel chemokine-binding protein decreased eosinophil influx and greatly ameliorated colitis, suggesting that strategies that interfere with the recruitment of eosinophils may be useful as therapy for colitis.

Cutting edge: serotonin is a chemotactic factor for eosinophils and functions additively with eotaxin.

Elevated levels of serotonin (5-hydroxytryptamine, 5-HT) are observed in the serum of asthmatics. Herein, we demonstrate that 5-HT functions independently as an eosinophil chemoattractant that acts additively with eotaxin. 5-HT2A receptor antagonists (including MDL-100907 and cyproheptadine (CYP)) were found to inhibit 5-HT-induced, but not eotaxin-induced migration. Intravital microscopy studies revealed that eosinophils roll in response to 5-HT in venules under conditions of physiological shear stress, which could be blocked by pretreating eosinophils with CYP. OVA-induced pulmonary eosinophilia in wild-type mice was significantly inhibited using CYP alone and maximally in combination with a CCR3 receptor antagonist. Interestingly, OVA-induced pulmonary eosinophilia in eotaxin-knockout (Eot-/-) mice was inhibited by treatment with the 5-HT2A but not CCR3 receptor antagonist. These results suggest that 5-HT is a potent eosinophil-active chemoattractant that can function additively with eotaxin and a dual CCR3/5-HT2A receptor antagonist may be more effective in blocking allergen-induced eosinophil recruitment.

Lysophosphatidic acid induces chemotaxis, oxygen radical production, CD11b up-regulation, Ca2+ mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator, which is generated by secretory type II phospholipase A(2) and is thought to play a major role in the pathogenesis of atopic diseases. In this study, the biological activity of LPA on human eosinophils was characterized. We showed by reverse transcription and PCR that human eosinophils express the mRNA of the LPA receptors endothelial differentiation gene (EDG)-2 and EDG-7. Experiments revealed that LPA has chemotactic activity toward eosinophils, stimulates the production of reactive oxygen metabolites, and induces up-regulation of the integrin CD11b. Signal pathway measurements indicated Ca(2+)-mobilization from intracellular stores and transient actin polymerization upon stimulation with LPA. Cell responses elicited by LPA were inhibited by pertussis toxin indicating that in eosinophils the LPA receptor(s), presumably EDG-2 and/or EDG-7, are coupled to G(i/o) proteins. Moreover, LPA-induced activation of eosinophils could be completely blocked by the EDG-2/EDG-7 antagonist diacylglycerol pyrophosphate. In addition, at optimal doses the changes induced by LPA were comparable to those obtained by the other well-characterized chemotaxins. These results indicate that LPA is a strong chemotaxin and activator of eosinophils. These findings point to a novel role of LPA in the pathogenesis of diseases with eosinophilic inflammation such as atopic diseases as chemotaxin as well as activator of proinflammatory effector functions.

TGF-beta and IL-13 synergistically increase eotaxin-1 production in human airway fibroblasts.

Chronic diseases may involve an "innate" response followed by an adaptive immune response, of a Th1 or Th2 variety. Little is known regarding the interactions of these responses. We hypothesized that TGF-beta1 (innate response factor associated with wound repair) in combination with IL-13 (Th2 factor) might augment inflammatory processes associated with asthma. Airway fibroblasts were cultured from asthmatic subjects and normal controls. These fibroblasts were exposed to TGF-beta1 and IL-13 alone or in combination, and eotaxin-1 expression and production were evaluated. At 48 h, eotaxin-1 production was markedly increased with the combination of TGF-beta1 and IL-13 (p < 0.0001) compared with either stimulus alone. mRNA increased slightly at 1 h with IL-13 or TGF-beta1 plus IL13, peaked, and became significantly increased over IL-13 alone at 24 h. Protein was measurable from 6 h with IL-13 and TGF-beta1 plus IL-13, but greater levels were measured over time with the combination. Actinomycin ablated the increase in mRNA and protein seen with IL-13 alone and with TGF-beta1 plus IL-13. Cycloheximide blocked the increase in mRNA at 6 h in both conditions, but also blocked the increase at 24 h with TGF-beta1 plus IL-13. STAT-6 was rapidly activated with both IL-13 and the combination, without difference. Finally, eotaxin-1-positive fibroblasts were identified in severe asthma biopsies in greater numbers than in normals. These results support the concept that interactions of innate and adaptive immune systems may be important in promoting the tissue eosinophilia of asthma, particularly in those with more severe disease.

Effects of an eosinophil chemotaxis inhibitor, TAK-661, on antigen-induced asthmatic responses in allergic sheep.

Eosinophils have been shown to play a role in allergen-induced airway responses. The aim in this study was to examine the effects of TAK-661, a newly developed product as a specific inhibitory agent of eosinophil chemotaxis, on antigen-induced asthmatic responses in allergic sheep model. Seven Ascaris-sensitive, "dual-respondent" allergic sheep were provocated by an Ascaris suum antigen or phosphate-buffered saline 2 hrs after intra-stomach administration of TAK-661 or a placebo. Pulmonary resistances were measured throughout the experiment, and airway responsiveness to methacholine, bronchoalveolar lavage (BAL), and histological examination were performed 8 hrs after the antigen challenge. Antigen provocation induced dual-phase bronchoconstriction, eosinophilia in BAL and eosinophil infiltration into the airway wall, and an increase in airway responsiveness in placebo-treated sheep. The administration of TAK-661 significantly reduced the bronchoconstriction during the late phase, along with the inhibition of eosinophilia in BAL and the eosinophil infiltration into the airway wall. TAK-661 had a tendency to reduce early-phase bronchoconstriction and airway hyperresponsiveness, but there were no significant differences. These findings suggest that the eosinophil accumulation into the airway induced by antigen provocation may contribute to the development of late-phase bronchoconstriction, however, the development of airway hyperresponsiveness during late asthmatic response may not always be due to only eosinophilic inflammation in the airway.

Eotaxin and asthma.

Eotaxin is a small protein that is produced in the lungs of asthmatic patients and is a potent chemoattractant for eosinophils. Eotaxin, a CC chemokine, stimulates the migration of eosinophils from the small blood vessels in the lungs by acting on the CC chemokine receptor CCR3, which is located on the leukocyte cell surface. In the past year, three low molecular weight compounds have been developed that can block this receptor. Such compounds may be developed into orally available drugs aimed at preventing eosinophil recruitment and, hence, the pathogenesis associated with the activation of these cells within the lung tissue.

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 B(4), 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 approximately 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.

Bleomycin stimulates lung fibroblast and epithelial cell lines to release eosinophil chemotactic activity.

The presence of eosinophils in the lungs of patients with pulmonary fibrosis correlates with poor prognosis or resistance to therapy. Furthermore, eosinophils localize to areas undergoing active fibrosis. It was hypothesized that a human lung fibroblast (HFL-1) and a human lung epithelial cell line (BEAS-2B) might release eosinophil chemotactic activity (ECA) in response to bleomycin, a chemotherapeutic agent associated with pulmonary fibrosis. HFL-1 and BEAS-2B cells were cultured in the presence of bleomycin and their supernatant fluids evaluated for ECA by means of a Boyden chamber method. HFL-1 and BEAS-2B cells released ECA in a dose- and time-dependent manner in response to bleomycin, and partial characterization revealed that the ECA was heterogeneous. ECA release from HFL-1 and BEAS-2B cells was significantly reduced by a leukotriene B4 (LTB4) receptor antagonist and an antibody directed against granulocyte-macrophage colony-stimulating factor. HFL-1 cells released LTB4, eotaxin, and GM-CSF constitutively, and BEAS-2B cells released LTB4, eotaxin, regulated on activation, normal T-cell expressed and presumably secreted, and GM-CSF constitutively. In both cases, the release of GM-CSF was significantly increased in response to bleomycin. These data suggest that lung fibroblasts and epithelial cells may modulate eosinophil recruitment into the lung in bleomycin-induced pulmonary fibrosis.

Glucocorticosteroids inhibit mRNA expression for eotaxin, eotaxin-2, and monocyte-chemotactic protein-4 in human airway inflammation with eosinophilia.

How eosinophils are preferentially recruited to inflammatory sites remains elusive, but increasing evidence suggests that chemokines that bind to the CCR3 participate in this process. In this study, we investigated the transcript levels and chemotactic activity of CCR3-binding chemokines in nasal polyps, a disorder often showing prominent eosinophilia. We found that mRNA expression for eotaxin, eotaxin-2, and monocyte-chemotactic protein-4 was significantly increased in nasal polyps compared with turbinate mucosa from the same patients, or histologically normal nasal mucosa from control subjects. Interestingly, the novel CCR3-specific chemokine, eotaxin-2, showed the highest transcript levels. Consistent with these mRNA data, polyp tissue fluid exhibited strong chemotactic activity for eosinophils that was significantly inhibited by a blocking Ab against CCR3. When patients were treated systemically with glucocorticosteroids, the mRNA levels in the polyps were reduced to that found in turbinate mucosa for all chemokines. Together, these findings suggested an important role for CCR3-binding chemokines in eosinophil recruitment to nasal polyps. Such chemokines, therefore, most likely contribute significantly in the pathogenesis of eosinophil-related disorders; and the reduced chemokine expression observed after steroid treatment might reflect, at least in part, how steroids inhibit tissue accumulation of eosinophils.

Th1-derived cytokine IFN-gamma is a potent inhibitor of eotaxin synthesis in vitro.

Eotaxin potentially plays an integral role in tissue eosinophilia. Inasmuch as Th2-derived cytokine IL-4 has been shown to stimulate eotaxin generation, we investigated here the effect of Th1-derived cytokine IFN-gamma on human eotaxin production. IFN-gamma but not -alpha or -beta potently inhibited tumor necrosis factor (TNF)-alpha-induced eotaxin generation by dermal fibroblasts. The inhibitory effect was unique to eotaxin, because production of IL-8 or monocyte chemoattractant protein (MCP)-1 protein was not affected by the treatment with IFN-gamma. Furthermore, the suppressive effect of IFN-gamma was not cell-type or stimulus specific. The level of eotaxin mRNA increased within 2 h after activation with TNF-alpha and continued to increase up to 72 h. IFN-gamma did not inhibit, but rather augmented the TNF-alpha-induced accumulation of mRNA in the early phase ( approximately 6 h). However, in the later phase, IFN-gamma completely prevented the subsequent elevation of eotaxin mRNA and sustained it at low levels. Although the protective effect of IFN-gamma against allergic inflammation has been assumed to result from its sole regulation of the proliferation of Th2-type T lymphocytes, these results imply that IFN-gamma can also directly act on stromal cells to inhibit eotaxin production and consequently intervene in eosinophil recruitment.

CD26/dipeptidyl-peptidase IV down-regulates the eosinophil chemotactic potency, but not the anti-HIV activity of human eotaxin by affecting its interaction with CC chemokine receptor 3.

Chemokines attract and activate distinct sets of leukocytes. The CC chemokine eotaxin has been characterized as an important mediator in allergic reactions because it selectively attracts eosinophils, Th2 lymphocytes, and basophils. Human eotaxin has a penultimate proline, indicating that it might be a substrate for dipeptidyl-peptidase IV (CD26/DPP IV). In this study we demonstrate that eotaxin is efficiently cleaved by CD26/DPP IV and that the NH2-terminal truncation affects its biological activity. CD26/DPP IV-truncated eotaxin(3-74) showed reduced chemotactic activity for eosinophils and impaired binding and signaling properties through the CC chemokine receptor 3. Moreover, eotaxin(3-74) desensitized calcium signaling and inhibited chemotaxis toward intact eotaxin. In addition, HIV-2 infection of CC chemokine receptor 3-transfected cells was inhibited to a similar extent by eotaxin and eotaxin(3-74). Thus, CD26/DPP IV differently regulates the chemotactic and antiviral potencies of eotaxin by the removal of two NH2-terminal residues. This physiological processing may be an important down-regulatory mechanism, limiting eotaxin-mediated inflammatory responses.

Blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus.

Chemokines direct migration of immune cells into sites of inflammation and infection. Chemokine receptors are seven-transmembrane domain proteins that, in contrast to other cytokine receptors, cannot be easily engineered as soluble chemokine inhibitors. Poxviruses encode several soluble cytokine receptors to evade immune surveillance, providing new strategies for immune modulation. Here we show that vaccinia virus and other orthopoxviruses (cowpox and camelpox) express a secreted 35-kDa chemokine binding protein (vCKBP) with no sequence similarity to known cellular chemokine receptors. The vCKBP binds CC, but not CXC or C, chemokines with high affinity (Kd = 0.1-15 nM for different CC chemokines), blocks the interaction of chemokines with cellular receptors, and inhibits chemokine-induced elevation of intracellular calcium levels and cell migration in vitro, thus representing a soluble inhibitor that binds and sequesters chemokines. The potential of vCKBP as a therapeutic agent in vivo was illustrated in a guinea pig skin model by the blockade of eotaxin-induced eosinophil infiltration. a feature of allergic inflammatory reactions. Furthermore, vCKBP may enable the rational design of antagonists to neutralize pathogens that use chemokine receptors to initiate infection, such as HIV or the malarial parasite.

Identification of IL-5 and RANTES as the major eosinophil chemoattractants in the asthmatic lung.

The study was carried out to identify those molecules that are important in vivo in the attraction of eosinophil granulocytes to the lungs of patients with asthma. Asthmatic patients with birch pollen allergy had lavages performed before and during the pollen season, and the chemotactic activity of the bronchoalveolar lavage fluid was tested against normal eosinophils. The activity was significantly increased during the pollen season as compared with the activity before the pollen season (p less than 0.01). Neutralizing antibodies to IL-2, IL-5 and IL-8, leukemia inhibitory factor, and to RANTES were added to the bronchoalveolar lavage fluid. Antibodies to IL-5 and RANTES, but not to IL-2 and IL-8 or leukemia inhibitory factor, significantly inhibited the chemotactic activity for eosinophils (p less than 0.001). It is concluded that IL-5 and RANTES are important chemoattractants in the lungs of patients with allergic asthma. The effect of IL-5 may be that of a cofactor to the chemotactic molecules, of which RANTES may be one of the most important in allergic asthma.

[Inhibitory effects of cyclosporin A and FK-506 on eosinophil chemotactic factor activity in culture supernatants of mononuclear cells from asthmatics].

We have previously reported that the 5-day culture supernatants of peripheral blood mononuclear cells (PBMC) from Dermatophagides farinae (DF) sensitive asthmatics stimulated with 10 ng/ml DF antigen contain eosinophil chemotactic activity (ECA) with an apparent molecular weight greater than 30000 Da. In the present study, we examined the effects of CyA and FK on the ECA. ECA was tested using modified Boyden chamber method. We found that when CyA or FK was added to the culture throughout the experiment, the production of the factors with ECA by PBMC was inhibited in a dose-dependent manner. These inhibitory effects were unchanged by the addition of a sufficient dose of IL-2 to the culture medium. Isoelectrofocusing of the PBMC culture supernatants consistently yielded a major ECF activity at pH 7.0-7.5. The addition of CyA inhibited this major peak. In conclusion, these results suggest that mononuclear cells stimulated with related antigen produce substances which possess ECA and that CyA and FK can block the production of this substance. Therefore, there is a possibility that an immunosuppressive agent may be useful in bronchial asthma therapy by inhibiting the migration of eosinophils.

Interference of cetirizine with the late eosinophil accumulation induced by either PAF or compound 48/80.

1. The effect of topical or systemic treatment with the histamine H1-receptor antagonist, cetirizine, on the rat pleural eosinophil accumulation induced by PAF or compound 48/80 was investigated. The number of pleural resident eosinophils increased 6 h after the intrathoracic (i.t.) injection of PAF (1 microgram/cavity), peaked within 24 h and persisted significantly augmented for at least 96 h. Compound 48/80 (25 micrograms/cavity) also produced a long lasting pleural eosinophilia but this was first noted only 24 h after stimulation. 2. Intraperitoneal pretreatment with cetirizine inhibited eosinophilia induced by either PAF (ED50 = 19 mg kg-1) or compound 48/80 (ED50 = 14 mg kg-1) whereas meclizine, another histamine H1-receptor antagonist, was inactive. 3. Administered locally, cetirizine (5 and 15 micrograms/cavity) also dose-dependently inhibited both PAF- and compound 48/80-induced eosinophilia, providing evidence that its inhibitory effect is not due to any action upon circulating eosinophils. Consistent with this result, incubation of isolated peritoneal eosinophils with cetirizine failed to modify in vitro eosinophil migration caused by PAF. 4. Since the late eosinophilia induced by PAF may depend on the synthesis of a transferable protein mediator, cetirizine was administered to donor or recipient rats in order to determine whether it interferes with the generation or with the expression of this protein. We showed that only the treatment of recipient rats abolished the transfer of the eosinophilotactic activity, indicating that cetirizine does not modify the generation but inhibits the expression of this activity. 5. Our findings indicate that cetirizine is able to inhibit eosinophil accumulation by acting locally. The mechanism is neither related to its recognized ability to antagonize histamine H,-receptors nor to a direct action upon circulating eosinophils.

Studies on leukotriene D4 as an eosinophil chemoattractant.

The effects of LTD4 on eosinophil motility were studied in order to indicate its potential role as an eosinophil chemoattractant. The guinea pig was selected as a suitably sensitive species for in vivo studies. Eosinophil infiltration was quantified by digital image analysis of 6 microns paraffin ocular and cutaneous tissue sections stained by Luna's method. LTD4, applied topically to the ocular surface, caused pronounced eosinophil infiltration into the conjunctival epithelium and was more potent and efficacious than a variety of other putative mediators of allergy. Pretreatment with the LT-antagonist SK&F 104353 (i.v. 1 mg/kg) abolished LTD4-induced eosinophil infiltration into the conjunctiva. Eosinophil infiltration did not occur in other ocular anterior segments structures such as the cornea, iris and ciliary body after either topical application or intracameral injection. LTD4 did not cause eosinophil emigration into skin following intradermal injection, despite causing an increase in cutaneous microvascular permeability at identical doses. These studies indicate that LTD4 may cause eosinophil emigration in vivo according to tissue-dependent regulation.

The regulation of tissue eosinophilia. III. In vitro production of eosinophil-directed chemotactic inhibitory factor by T lymphocytes of complete Freund's adjuvant-treated guinea-pigs.

Guinea-pig spleen cells treated with complete Freund's adjuvant (CFA) produce eosinophil-directed chemotactic inhibitory factors (ECIF). The inhibition is selective for the response of eosinophils to delayed ECF-a, which had been isolated from 24-hr-old inflamed skin lesions induced by DNP-Ascaris extract in sensitized animals and had been confirmed as being a T lymphocyte-derived lymphokine. ECIF activity is absorbed by incubation with eosinophils, but not with macrophages or neutrophils. The cells spontaneously release ECIF; pretreatment with a protein synthesis inhibitor reduces ECIF production, indicating that protein synthesis is essential. The source of ECIF seems to be lymphocytes, probably T lymphocytes. ECIF activity is recovered in two separate fractions: one elutes near bovine serum albumin (MW 70,000) and the other near cytochrome c (MW 12,500). ECIF binds to peanut agglutinin- or Limulus polyphemus agglutinin-coupled agarose beads. Furthermore, ECIF activity is blocked when eosinophils are incubated with D-galactose or sialic acid. These results suggest that ECIF derived from T lymphocytes of CFA-treated animals modulate the delayed ECF-a-mediated tissue eosinophilia, and that terminal galactose and/or sialic acid residues are essential for ECIF activity.

The regulation of tissue eosinophil chemotactic factor and inhibitor in allergic skin lesions of Freund's complete adjuvant-treated guinea-pigs.

The regulation of tissue eosinophilia induced by dinitrophenyl-ascaris extract (DNP-As) was investigated in guinea-pigs. Biphasic tissue eosinophilia peaking at 6 and 24 hr was observed in the skin lesions in Bordetella pertussis vaccine (Bp)-treated animals. In contrast, only the early phase of tissue eosinophilia was observed in Freund's complete adjuvant (FCA)-treated animals. Although less eosinophil chemotactic activity was detected in 24-hr-old skin extract of FCA-treated animals (FCA-extract), evident activity was recovered in the concanavalin A eluate (Con A-eluate) when FCA-extract was fractionated by Con A Sepharose. The chemotactic factor in Con A-eluate of FCA-extract was confirmed to be the T cell-derived eosinophil chemotactic factor, termed Delayed ECF-a, which has been isolated from allergic skin lesions by immunoadsorption. Another factor from the same skin lesions, Delayed ECF-b (which is a serum-derived one), was not detected in the FCA-extract. When eosinophils were mixed or pretreated with Con A-effluent of FCA-extract, the treated cells failed to be attracted by Delayed ECF-a, while the response to Delayed ECF-b was not affected, indicating that the inhibition was selective for Delayed ECF-a but not for Delayed ECF-b, and the eosinophil chemotactic inhibitory factor (ECIF) acts on eosinophils directly. Major ECIF activity was associated with a mol. wt. of 70,000 and minor with 12,500. Furthermore, the activity was absorbed by eosinophils but not by macrophages suggesting that eosinophils have receptor sites for ECIF. It was thus suggested that the appearance of ECIF, which is selective for the response of eosinophils to Delayed ECF-a, and decreased Delayed ECF-b production resulted in the inhibition of delayed tissue eosinophilia in FCA-treated guinea-pigs.