Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases.

Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice.

The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNFalpha in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNFalpha in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and -challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNFalpha by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNFalpha or Ad/IL-4 into OVA-sensitized and -challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNFalpha and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.

CD40 expression by human peripheral blood eosinophils.

In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.

Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation.

Using adenoviral-mediated gene transfer techniques, the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue-directed expression of GM-CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM-CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL)-5, IL-6, macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others.

Helicobacter pylori-negative gastric and duodenal ulcers.

It is unclear whether Helicobacter pylori infection is essential to the development of peptic ulcers. In this study, we examined the rates of H. pylori-negativity among patients with peptic ulcers. We also attempted to clarify the characteristics of H. pylori-negative peptic ulcers to throw light on the pathogenesis of peptic ulcers. The study included 215 consecutive patients with gastric ulcers (GUs) and 120 consecutive patients with duodenal ulcers (DUs). After routine endoscopic examination and phenol red dye endoscopy, forceps biopsies were performed for culture, histology, and the rapid urease test. A patient was considered H. pylori-negative when the serum anti-H. pylori IgG and the three tests on biopsied specimens were all negative. H. pylori-negative rates were 3.2% in the patients with GUs and 1.7% in the patients with DUs. Lack of atrophy of the gastric mucosa was significantly more common in the H. pylori-negative patients with GUs. A history of ulcer disease was less common and antral ulcers were more common in H. pylori-negative GU patients, but not significantly so. As the urea breath test had not been performed, the possibility of a false-negative result cannot be completely ruled out, but we believe that the H. pylori-negative rate in our study is more reliable than these rates in previous reports, because we visualized H. pylori distribution by phenol red dye endoscopy to avoid false-negative results in biopsies, and we used both biopsy and serum anti-H. pylori IgG findings to establish an H. pylori-negative diagnosis. Since H. pylori-negative peptic ulcers certainly exist, H. pylori infection is thought not to be essential to the development of peptic ulcers. There were few differences between the characteristics of H. pylori-negative and H. pylori-positive peptic ulcers in our study. A large-scale study is required to clarify the characteristics of H. pylori-negative peptic ulcers.

Clinicopathologic and cytogenetic analyses of three cases of primary uterine non-Hodgkin's lymphoma.

Primary uterine non-Hodgkin's lymphoma (NHL) is an extremely rare disease. To accumulate more information on clinical data, we report three cases of primary uterine NHL with apparently the first demonstration of karyotypic analysis. Histological diagnosis was diffuse large B cell type in all patients. Two of them with advanced stage showed chemoresistance and a short survival. The remaining case with early stage showed an uneventful course following operation. No common chromosomal abnormality was detected. The therapeutic strategy for uterine NHL might therefore be similar to that for other types of aggressive NHL, although a larger study is needed.

[Clinical evaluation of panipenem/betamipron as a second line chemotherapy in severe infections associated with hematological disorders].

Thirty three patients with severe infections associated with hematological disorders were treated with panipenem/betamipron as a second line chemotherapy. Of these, 30 patients were evaluated for effectiveness. An excellent response was obtained in 14 patients (46.7%) and a good response in 5 (16.7%), and the overall efficacy rate was 63.3%. Efficacy rates were 3/6 in patients with sepsis, 68.4% (13/19) in patients with fever of undetermined origin, 2/4 in patients with pneumonia. In patients whose peripheral granulocyte count was below 100/microliter at the start of chemotherapy, the efficacy rate was 3/7. Side effects were observed in 5 of 33 patients (15.2%). These results show that PAPM/BP is useful as a second line chemotherapy for the treatment of severe infections in patients with hematological disorders.

[CD34-positive cell selection using an Isolex 300 system in patients with solid tumors and its application for autologous peripheral blood stem cell transplantation].

Using an Isolex 300 immunomagnetic cell separator, we carried out CD34+ cell selection in samples from 4 patients with solid tumors: 2 patients with relapsed breast cancer, 1 post-operative patient with advanced breast cancer, and 1 post-operative patient with advanced ovarian cancer. Peripheral blood stem cells were mobilized by G-CSF and high-dose chemotherapy (CAF or VIC-E regimen). The mean recovery rate for CD34+ cells was 62.0% and the mean purity was 89.5%. However, the mean recovery for colony-forming cells (CFC) was only 10.9%, suggesting that recovered CD34+ cells may be damaged during the separation of immunomagnetic beads by releasing peptide or by 4 cycles of cytocentrifugation (at 800 G for 10 min). Approximately 30% of the CFC, consisting largely of BFU-E, had been recovered in the CD34- cell fraction. Recently, it has been reported that primitive long-term hematopoietic repopulating cells may express weakly or not at all for CD34 antigen. This suggests that careful follow-up monitoring is necessary for long-term hematopoietic reconstitution after CD34+ cell transplantation.

[Comparison of CRH test and ACTH test in patients with bronchial asthma].

Although glucocorticoid is the most effective agent for bronchial asthma, its systemic administration leads to suppression of adrenocortical function. Rapid ACTH test has been performed for assessing the function of the hypothalamic-pituitary-adrenocortical (HPA) system of asthmatics. Recently human corticotropin-releasing hormone (CRH) has been chemically synthesized. In order to evaluate clinical usefulness of CRH, we compared CRH test with ACTH test in 17 patients with bronchial asthma (3 patients out of them concurrently receiving prednisolone 5-10 mg/day). Both tests were carried out within 2 weeks after 6 month treatment with fluticasone propionate (800 micrograms/day) inhaled via pMDI. There is no significant difference between results obtained from the both tests. Thus, dividing subjects into high and low responders based on an extent of increases in plasma ACTH levels after the CRH injection, we found a significant difference in maximal plasma concentrations of cortisol between after CRH and ACTH injections in the low responders. Therefore, in some patients, CRH test provides more accurate assessment of the function of HPA system than ACTH test.

[Non-real-time computed tomography-guided percutaneous ethanol injection therapy for hepatocellular carcinoma undetectable by ultrasonography].

The purpose of this study was to evaluate the feasibility of non-real-time CT-guided percutaneous ethanol injection therapy (PEIT) for hepatocellular carcinoma (HCC, 37 lesions) untreatable by ultrasonography-guided (US)-PEIT. The HCC lesion was localized on the lipiodol CT image with a graduated grid system. We advanced a 21 G or 22 G needle in a stepwise fashion with intermittent localization scans using a tandem method to position the tip of the needle in the lesion. Ethanol containing contrast medium was injected with monitoring scans obtained after incremental volumes of injection, until perfusion of the lesion was judged to be complete. A total of 44 CT-PEIT procedures were performed. The average number of needle passes from the skin to the liver in each CT-PEIT procedure was 2.3, the average amount of ethanol injected was 14.4 ml, and the average time required was 49.3 minutes. Complete perfusion of the lesion by ethanol on monitoring CT images was achieved in all lesions with only a single or double CT-PEIT procedure without severe complication. Local recurrence was detected only in 5 lesions. At present, it is more time-consuming to perform CT-PEIT than US-PEIT because conventional CT guidance is not real-time imaging. However, it is expected that this limitation of CT-PEIT will be overcome in the near future with the introduction of CT fluoroscopy. In conclusion, CT-PEIT should prove to be a feasible, acceptable treatment for challenging cases of HCC undetectable by US.

The involvement of type 1a angiotensin II receptors in the regulation of airway inflammation in a murine model of allergic asthma.

BACKGROUND: There has been increasing evidence suggesting the involvement of angiotensin II (Ang II) and type 1 Ang II receptors (AT1) in the pathogenesis of bronchial asthma. However, whether such an involvement would promote or suppress the pathophysiology of asthma is controversial. OBJECTIVE: The aim of this study was to investigate the role of AT1 in the development of allergic airway inflammation. METHODS: Agtr1a+/+ [wild-type C57BL/6 mice (WT)] and Agtr1a-/- mice [AT1a knockout mice (AT1aKO)] with a genetic background of C57BL/6 were systemically sensitized to ovalbumin (OVA), followed by OVA inhalation. OVA-specific IgE in serum obtained just before the inhalation was measured. Bronchoalveolar lavage (BAL) fluid and lung tissues were obtained at various time-points. Cell numbers and differentiation, and cytokine contents in BAL fluids were determined. Peribronchial accumulation of eosinophils and mucus inclusions in the bronchial epithelium were evaluated in lung tissues stained histochemically. Cell numbers and differentiation in BAL fluids of the mice were also determined after lipopolysaccharide (LPS) inhalation. RESULTS: The levels of OVA-specific IgE in AT1aKO were significantly higher than those in WT. The numbers of total cell, eosinophils and lymphocytes in BAL fluids 7 days after OVA inhalation in AT1aKO were significantly higher than those in WT. Airway inflammation in bronchial tissues in terms of eosinophil accumulation and mucus hypersecretion in AT1aKO was also stronger than in WT. The contents of IL-4, IL-5 and IL-13, but not IFN-gamma, in BAL fluids of AT1aKO were significantly higher than those of WT. In contrast, neutrophil accumulation in BAL fluids after LPS inhalation was significantly higher in WT than in AT1aKO. CONCLUSION: AT1a might be involved in the negative regulation of the development of allergic airway inflammation through polarizing the T-helper (Th) balance towards Th1 predominance. Therefore, it would be of clinical importance to investigate the effects of long-term administration of AT1 blockers on the Th1/Th2 balance in hypertensive patients with bronchial asthma.

[Expression of adhesion molecules in bronchial asthma].

Eosinophilic infiltration into the airway is thought to be a key process to induce late asthmatic response, and it seems to be very important for the treatment of bronchial asthma to study the precise mechanisms of selective infiltration of eosinophils. In this study, we examined which adhesion molecules were involved in selective eosinophil infiltration into the airway, by immunohistochemistry, immuno-electron microscopy and in situ hybridization methods. In the sputum and peripheral blood eosinophils of asthmatics, Mac-1 was strongly expressed and LFA-1, VLA-4 and sLe-X were also expressed. In the bronchial submucosa of lung tissues from autopsy and biopsy of patients with bronchial asthma, immunoreactivity of ICAM-1 was detected in the endothelial cells, the basal layer of the bronchial epithelium, mononuclear cells and extracellular spaces, and VCAM-1 was also detected in the endothelial cells, but ELAM-1 was weakly detected. In addition, immunoreactivities of Mac-1, LFA-1, and VLA-4 were detected in eosinophils infiltrated into the bronchial submucosa, but sLe-X was weakly detected. These results suggest that binding between ICAM-1 and Mac-1 or LFA-1, VCAM-1 and VLA-4, not but ELAM-1 and sLe-X, is mainly involved in eosinophil infiltration into the airway in allergic reaction such as bronchial asthma.

Activation and transforming growth factor-beta production in eosinophils by hyaluronan.

To investigate whether extracellular matrix glycosaminoglycan hyaluronan (HA) modulates eosinophil activation and transforming growth factor (TGF)-beta production by eosinophils, human peripheral blood eosinophils (purity > 99%) from 12 patients with mild to moderate asthma or six healthy subjects were isolated and incubated with increasing concentrations of low molecular weight (mol wt) HA ( approximately 0.2 x 10(6) D) or high mol wt HA (3.0 to approximately 5.8 x 10(6) D). We found that the low mol wt HA has a pronounced effect on eosinophil survival in both patients with asthma and healthy subjects in a dose-dependent fashion on Days 2 and 4. Whereas the high mol wt HA had a smaller effect on eosinophil survival than did the low mol wt HA. The HA-mediated eosinophil survival was partially but significantly inhibited ( approximately 50% inhibition) by a blocking monoclonal antibody for CD44, a specific receptor of HA, and largely inhibited by an anti-granulocyte macrophage colony-stimulating factor (GM-CSF) neutralizing antibody but not by an anti-interleukin (IL)-3 or anti-IL-5 neutralizing antibody. In addition, the low mol wt HA increased GM-CSF messenger RNA (mRNA) expression and protein secretion by eosinophils in a dose-dependent fashion, suggesting that the HA-mediated eosinophil survival is due mainly to induction of GM-CSF release through partial CD44 signaling. Furthermore, we demonstrated that the low mol wt HA results in morphologic changes in eosinophils such as transforming from a round to a spindle shape and in homotypic aggregation, upregulates intercellular adhesion molecule-1 expression, and increases TGF-beta mRNA expression and protein secretion by eosinophils. These observations suggest previously unforeseen interactions between eosinophils and low mol wt extracellular matrix and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation and airway remodeling.

Potential role of interleukin-1 in allergen-induced late asthmatic reactions in guinea pigs: suppressive effect of interleukin-1 receptor antagonist on late asthmatic reaction.

Interleukin (IL)-1 is a pluripotential proinflammatory cytokine and is thought to be involved in the pathogenesis of bronchial asthma and late asthmatic reactions (LARs). To determine whether IL-1 plays a role in LAR, guinea pigs sensitized with Ascaris antigen were used. We evaluated IL-1 production by immunostaining with anti-IL-1 beta antibody and elucidated the action of IL-1 in LAR with recombinant IL-1 receptor antagonist. Immunostaining revealed that IL-1 beta-like immunoreactivity-positive cells increased in the airway walls and in bronchoalveolar lavage fluid after the antigen challenge. IL-1 receptor antagonist protein pretreatment reduced the generation of LAR in terms of pulmonary resistance. IL-1 receptor antagonist protein pretreatment did not change cellular components but reduced the percentage of hypodense eosinophils in bronchoalveolar lavage fluid. We also studied the direct effect of recombinant human IL-1 beta on pulmonary resistance and eosinophil activity measured as released eosinophil peroxidase activity. Recombinant human IL-1 beta did not change pulmonary resistance but primed eosinophils to release eosinophil peroxidase activity in response to platelet activating factor. Therefore these results suggest that IL-1 was produced in sensitized pulmonary tissue of guinea pigs by allergen exposure and played a role in the generation of LAR, at least partially by modulating the activation of eosinophils.

Apically localized IP3 receptors control chloride current in airway gland acinar cells.

We examined the role of inositol 1,4,5-trisphosphate (IP3) receptors in acetylcholine (ACh)-induced chloride (Cl-) current in acinar cells of human and feline airway submucosal glands, using whole cell patch-clamp analysis. ACh (10 nM-1 microM) induced an initial Cl- current followed by a K+ current, and lower doses of ACh (1-10 nM) often induced oscillations of both currents, which were mimicked by the application of intracellular IP3. Neither isoproterenol (-10 microM) nor raising intracellular adenosine 3,5-cyclic monophosphate induced any current. Caffeine (20-50 mM) and intracellular ryanodine (1-100 microM) induced a K+ current alone without Cl- current. Monoclonal antibodies to the IP3 receptor abolished both ACh-induced K+ and Cl- currents. Immunohistochemical analysis revealed the localization of IP3 receptors on both the cytosol and some regions of the endoplasmic reticulum beneath the apical membrane of acinar cells. These results indicate that apically localized IP3 receptors control Cl- secretion from airway submucosal gland cells.

TNF-alpha mRNA expression in diaphragm muscle after endotoxin administration.

We studied gene expression and production of TNF-alpha in the diaphragm tissue and changes of muscle contractile properties after endotoxin injection (Escherichia coli, 20 mg/kg) in 88 rats. We assessed the muscle contractile properties by force-frequency curves and twitch kinetics using dissected diaphragm muscle strips. The peak tensions of force-frequency curves decreased from control values (2.15 +/- 0.2 kg/cm2) up to 4 h (0.81 +/- 0.17, p < 0.001), and then increased at 6 h (1.36 +/- 0.19, p < 0.05) after endotoxin injection. The cytotoxic activity on L929 cells in arterial blood samples maximally increased at 2 h (p < 0.001), then decreased to 6 h (p < 0.05). TNF-alpha mRNA in diaphragm tissue was detected by Northern blot method at 1 and 1.5 h, and the immunolocalization of TNF-alpha was evaluated at 2 and 4 h by immunohistochemistry in the muscle tissues. Furthermore, preinjection with anti-m TNF-alpha antibody prevented the decrement of force-frequency curves after endotoxin injection of 10 microliters/kg. From this evidence that TNF-alpha gene expression and production occurred in the diaphragm tissue, but anti-m TNF-alpha antibody preinjection prevented the deterioration of diaphragm muscle contractile properties, we suggest that TNF-alpha may act on muscle cells extracellularly.

Production of tumor necrosis factor with IgE receptor triggering from sensitized lung tissue.

Mast cells and basophils have been known to play a central role in allergic inflammation through the release of chemical mediators by cross-linkage of IgE receptors. The IgE receptor triggering and calcium ionophore A23187 have also been shown to induce gene expression and production of tumor necrosis factor (TNF) by rat basophilic leukemia cells. In the present study, we examined whether IgE receptor triggering could induce gene expression and production of TNF in rat lung tissue. The lung tissue released not only histamine but also cytotoxic activity on L929 cells 2 and 4 h after incubation with dinitrophenyl conjugated to ovalbumin (DNP-OVA) following passive sensitization with anti-DNP monoclonal rat IgE antibody, whereas neither DNP-OVA nor anti-DNP IgE antibody could induce the cytotoxic activity when used solely. Calcium ionophore A23187 also could induce both histamine release and cytotoxic activity. These activities induced by IgE receptor triggering, A23187, and lipopolysaccharide were completely neutralized by preincubation with anti-mouse TNF-rabbit serum, but not with normal rabbit serum. Northern blot analysis using cDNA probe of mouse TNF demonstrated expression of TNF gene as early as 2 h after IgE receptor triggering. These data demonstrating that IgE receptor triggering induced gene expression and production of TNF in lung tissue suggest the participation of TNF in the pathogenesis of late asthmatic response through its biologic activities such as the attraction and activation of neutrophils and eosinophils.

Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia.

Interleukin-10 (IL-10) is a potent anti-inflammatory/immune cytokine and has received growing attention for its therapeutic potential. To aid therapeutic studies of IL-10 in vivo, a replication-deficient adenoviral vector expressing mouse IL-10 was constructed and characterized. The transgene protein IL-10 was shown to markedly inhibit endotoxin-induced tumor necrosis factor alpha (TNF alpha) production by mouse and rat macrophages in vitro. Intramuscular injection of this vector in mice resulted in efficient expression of transgene mRNA in the muscle and active release of IL-10 protein into the bloodstream. To investigate the therapeutic potential of IL-10 using this vector, endotoxemia was induced by intraperitoneal injection of a sublethal dose of endotoxin. Expression of TNF alpha and IL-6 mRNA in the lung, spleen and heart and the circulating levels of these cytokines markedly increased in endotoxemia. This endotoxin-induced TNF alpha and IL-6 up-regulation was however suppressed in mice expressing IL-10 after intramuscular gene transfer. While cytokine gene expression was inhibited to varying degrees in different organs, a maximal reduction was seen in the lung, thus also indicating the efficacy of systemic IL-10 gene product at multiple tissue sites. Finally, we provided evidence that only when present in abnormally high concentrations in the circulation following intraperitoneal gene delivery, IL-10 by itself had some toxic effects of transient nature, primarily manifested by acute phase reaction and hemostatic disturbance. Thus, our studies demonstrate the usefulness of adenoviral vectors for therapeutic applications of IL-10 in vivo.

Effect of whole-body x-irradiation on antigen-induced airway response in sensitized guinea pigs.

The objectives of this study were to test the hypothesis that x-irradiation inhibits the late asthmatic response (LAR) without influencing the early asthmatic response (EAR) and to examine the mechanism of the inhibitory effect. Twenty sensitized guinea pigs were irradiated at a dose of 8 Gy. The next day, one-half of the animals were injected intravenously with spleen cells (2 x 10(8)) collected from unirradiated sensitized guinea pigs, whilst the other half were injected with vehicle only. Ten additional unirradiated sensitized guinea pigs also received vehicle only. Antigen inhalation challenge took place two days later. Pulmonary resistance was measured for 6 h after antigen exposure, and bronchoalveolar lavage and lung fixation were then undertaken. The area under the percentage pulmonary resistance curve 2-6 h after allergen inhalation was used for analysis of the LAR, while the maximal percentage change in pulmonary resistance was used for analysis of the EAR. Irradiation abolished the LAR (364.4+/-49.4 versus 62.8+/-10.4) without inhibiting the EAR (229.3+/-27.2 versus 278.7+/-40.2) and significantly inhibited the accumulation of eosinophils and lymphocytes in the airways. Transfer of spleen cells restored the LAR (334.4+/-66.8) and the recruitment of cells to the levels seen in unirradiated sensitized guinea pigs. In addition, transfer of only CD4+ T-lymphocytes separated from the spleen cells restored the LAR (439.4+/-62.1) and the cell infiltration into the airways. These inhibitory effects of x-irradiation were due to decreases in numbers of CD4+ T-lymphocytes.