Respiratory syncytial virus-infected pulmonary epithelial cells induce eosinophil degranulation by a CD18-mediated mechanism

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the beta2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.     

Multiple roles of interferon-gamma in the mediation of interleukin 12-induced tumor regression

Administration of recombinant interleukin 12 (IL-12) induces tumor regression that is associated with T-cell infiltration in the OV-HM ovarian carcinoma and CSA1M fibrosarcoma models. After confirming the blocking of regression by injection of anti-IFN-gamma monoclonal antibody (mAb), we investigated the mechanisms underlying the requirement of IFN-gamma in T-cell migration and tumor regression. T-cell migration was inhibited by injection of anti-IFN-gamma mAb to OV-HM tumor-bearing mice prior to IL-12 treatment. We examined, using the lymphoid cell migration assay, whether IFN-gamma is required for enhancing the migratory capacity of T cells or the T cell-accepting potential of tumor masses during IL-12 treatment. Spleen cells from IL-12-treated or untreated OV-HM-bearing mice were stained in vitro with a fluorescein chemical and transferred i.v. into OV-HM-bearing mice that were not treated with IL-12. Migration of donor cells was quantitated by counting the number of fluorescent cells on cryostat sections of tumor masses from recipient mice. Compared to spleen cells from OV-HM-bearing mice that were not treated with IL-12, enhanced migration was observed for cells from IL-12-treated OV-HM-bearing mice. Anti-IFN-gamma pretreatment of donor mice before IL-12 treatment did not reduce the migratory capacity of T cells, whereas migration was markedly inhibited in recipient mice injected with anti-IFN-gamma. Anti-IFN-gamma pretreatment decreased vascular cell adhesion molecule-1 (VCAM-1)-/intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels at tumor sites. Consistent with this, migration was also inhibited by treatment of recipient mice with either anti-VCAM-1 or anti-ICAM-1 mAb. In contrast to the OV-HM model, T-cell migration was not affected in the CSA1M model following preinjection of anti-IFN-gamma mAb. In this model, VCAM-1-/ICAM-1-positive blood vessels existed even after anti-IFN-gamma treatment, although tumor regression was completely inhibited. These results indicate that IFN-gamma plays two distinct roles in expressing the antitumor efficacy of IL-12: one is to support the T-cell acceptability of tumor masses, and the other is to mediate the antitumor effects of migrated T cells.     

Efficacy of anti-intercellular adhesion molecule-1 immunotherapy on immune responses to allogeneic hepatocytes in mice

Adhesion molecules appear to play important roles in vascularized organ allograft rejection, because antibodies directed against them are effective in prolonging survival of vascularized organ allografts in rodents. However, the efficacy of these agents for cellular allografts is unknown. The current studies were undertaken to determine the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on host immune responses to purified hepatocytes. Host mice (C3H, H-2(k)) grafted with hepatocytes in sponge matrix allografts (HC-SMA) received IgG isotype control, anti-ICAM-1, or anti-VCAM-1 monoclonal antibody (mAb) on days 0 through 9 after grafting. Twelve to 14 days later, host cells infiltrating the HC-SMA were assessed for the development of allospecific cytolytic T cells (allo-CTLs). Treatment with anti-ICAM-1 or anti-VCAM-1 mAb resulted in significantly decreased recruitment of host cells into HC-SMA (P < .035). However, only anti-ICAM-1 mAb resulted in abrogation of development of allo-CTLs in HC-SMA (P = .001). C3H (H-2(k)) hosts grafted with allogeneic hepatocytes from control C57BL/6 (H-2(b)) or ICAM-1 knockout [H-2(b)] mice elicited the development of allo-CTLs in HC-SMA (P = not significant). Furthermore, there was no difference in the development of allo-CTLs in HC-SMA of control hosts [C57BL/6, H-2(b)] compared with ICAM-1 knockout hosts (H-2(b)) (P = not significant). Treatment with anti-ICAM-1 mAb had no effect on the development of allo-CTLs in ICAM-1 knockout (H-2(b)) hosts bearing HC-SMA. The immunosuppressive effect of host treatment with anti-ICAM-1 mAb does not appear to be a consequence of simple blockage of donor hepatocyte or host immune cell expression of ICAM-1, but suggests a potential inhibitory effect on host immune cell activation or function, as well as an effect on recruitment of host cells to the allograft.     

Sequence analysis of the coat protein and 3''-noncoding region for Korean and other strains of sweet potato feathery mottle virus

A murine model of asthma is described in which we examined the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of airway reactivity, pulmonary eosinophilia, and inflammation. We sensitized wild-type control [C57BL/6J, (+/+)] and ICAM-1 knockout [C57BL/6J-ICAM-1, (-/-)] mice to ovalbumin (OVA), and challenged them with OVA delivered by aerosol (OVA-OVA) to induce a phenotype consistent with an asthmatic response. Bronchial responsiveness to methacholine and counts of cell numbers and measurements of eosinophil content and cytokine levels in bronchoalveolar lavage fluid (BALF) were significantly attenuated in ICAM-1(-/-) mice as compared with (+/+) mice. We also showed that the absence of ICAM-1 had no significant affects on the production of serum IgE antibody, but did have an effect on ex vivo lymphocyte proliferation. Additionally, immunohistochemistry: (1) revealed increased staining for vascular cell adhesion molecule-1 (VCAM-1) after antigen challenge in the ICAM-1(-/-) mice but not in the ICAM-1(+/+) controls; and (2) confirmed the presence of alternatively spliced forms of ICAM-1 in the lungs of ICAM-1(-/-) mice. Thus, despite the availability of alternate adhesion pathways in ICAM-1(-/-) mice, the absence of ICAM-1 prevented eosinophils from entering the airways. In summary, we found that the ICAM-1 knockout mice exhibited a significantly inhibited response to aerosol antigen challenge for most of the parameters examined, and conclude that ICAM-1 is an important ligand mediating T-cell proliferation in response to antigen, eosinophil migration into the airways, and the development of airway hyperreactivity (AHR) in allergen-sensitized and -challenged mice.     

Expression of intercellular adhesion molecule-1 in mice with Pseudomonas-induced pyelonephritis

PURPOSE: To investigate the role of intercellular adhesion molecule-1 (ICAM-1) in the renal inflammatory process, we studied the time-course fluctuation of ICAM-1 expression on inflammatory lesions in mice with experimentally induced bacterial pyelonephritis and the effect of in vivo administration of an anti-ICAM-1 monoclonal antibody (mAb) on leukocytic migration. MATERIALS AND METHODS: Ascending pyelonephritis was induced by transurethral instillation of Pseudomonas aeruginosa, and the expression of ICAM-1 in the pyelonephritic lesions was studied by immunohistochemical methods. RESULTS: The expression of ICAM-1 on the pyelonephritic lesions closely paralleled the degree of infiltration of neutrophils and macrophages until 3 days after infection. At 7 days after infection, though the degree of infiltration of these cells was quite high, expression of ICAM-1 was reduced. Treatment with the anti-ICAM-1 mAb in mice with bacterial pyelonephritis resulted in suppression of influx of neutrophils and macrophages in the infected sites until 3 days after infection. However, at 7 days after infection inhibition of the influx of these cells was not seen. CONCLUSIONS: These results suggest that ICAM-1 expression is transient and plays a key role in the influx of neutrophils and macrophages associated with the early-phase response, and that in the late phase ICAM-1 independent adhesion molecules may be more predominant.     

Induction of T-cell hyporesponsiveness by intrahepatic modulation of donor antigen-presenting cells

In this study, we examined the ability of varying populations of donor cells from B6 mice to induce hyporesponsiveness in T lymphocytes from C3H mice in vitro and in vivo. Small, resting B lymphocytes were inefficient stimulators of T-lymphocyte proliferation compared to splenic mononuclear cells (SMNC) and lipopolysaccharide (LPS)-induced B-cell blasts in vitro (P < 0.05). Pretreatment of SMNC with anti-B7-1 or anti-intracellular adhesion molecule-1 (ICAM-1) monoclonal antibodies (mAb) similarly resulted in inefficient stimulation of T-cell proliferation in vitro (P < 0.05). However, in vivo, only intrahepatic, but not intravenous, injection of donor cells into C3H mice resulted in decreased T-lymphocyte proliferation in response to restimulation by alloantigen. This effect was most pronounced following intrahepatic injection of resting B lymphocytes or SMNC pretreated with anti-ICAM-1 mAb compared to uninjected or intravenously injected mice (P < 0.05). The hyporesponsiveness was associated with an increased production of interleukin-4 (IL-4) by the responder T lymphocytes and correlated with enhanced skin allograft survival. These data demonstrate that intrahepatic injection of donor-derived cells induces T-lymphocyte hyporesponsiveness. The mechanism appears to be modulated by an ICAM-1-mediated signal resulting in expansion of an IL-4-producing T-lymphocyte population.     

Vascular endothelial cell expression of ICAM-1 and VCAM-1 at the onset of eliciting contact hypersensitivity in mice: evidence for a dominant role of TNF-alpha

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-alpha antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1alpha and IL-1beta had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-alpha, anti-IL-1alpha, and IL-1beta was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-alpha alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against alpha4 and beta2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-alpha and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-alpha.     

ICAM-1-dependent pathway is not critically involved in the inflammatory process of autoimmune encephalomyelitis or in cytokine-induced inflammation of the central nervous system

To determine whether the rat homolog of intercellular adhesion molecule 1 (ICAM-1) plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) we examined the effect of anti-ICAM-1 mAb, 1A29, on both active and passive EAE. We also examined its effect on a model of cytokine-induced inflammation in the central nervous system. Treatment of recipients of EAE effector cells with anti-ICAM-1 had no inhibitory activity, and in fact at high doses, treatment enhanced disease as evidenced by an earlier onset of symptoms. Treatment of active EAE with anti-ICAM-1 beginning on the day of sensitization did protect a proportion of animals from development of disease as well as reduce the severity of clinical signs in those which developed symptoms. Lymphocytes from both the draining lymph nodes and spleens of myelin basic protein (MBP)-immunized rats treated with anti-ICAM-1 failed to proliferate in response to MBP in vitro, suggesting that the antibody had prevented the animals from becoming sensitized to the antigen. Microinjection of tumor necrosis factor (TNF)-alpha into the spinal cords of rats led to the expression of ICAM-1 on vascular endothelium, and to the accumulation of leukocytes at sites of injection. The peak expression of ICAM-1 by endothelium and the peak accumulation of leukocytes following TNF alpha injection were not positively correlated. Furthermore, treatment of TNF alpha injected rats with anti-ICAM-1 did not inhibit the accumulation of leukocytes at the site of cytokine injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eosinophil recruitment to the lung in a murine model of allergic inflammation. The role of T cells, chemokines, and adhesion receptors

Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.     

The role of microglia and tumor-primed lymphocytes in the interaction between T lymphocytes and brain endothelial cells

We investigated the role of IFN-gamma activated microglia in the passage of T lymphocytes across a monolayer of brain endothelial cells (EC) in vitro. Microglia isolated from Fisher 344 (F344) newborn rats were stimulated with IFN-gamma (100 U/ml) for 48 h. T lymphocytes primed with glioma cells were 51Cr-labeled, and added to the monolayer of F344 brain EC. In the adhesion assay, when EC were cultured in medium containing the supernatant of reactive microglia before the assay was carried out, the number of T lymphocytes adhering was increased. In addition, this adhesion was blocked by the addition of anti-ICAM-1 mAb to the EC. In the migration assay, performed using the double chamber system, when reactive microglia adhered to the other side of EC, the number of T lymphocytes migrating to the underwell was also increased. When T lymphocytes were primed to tumor cells in vivo, both their adhesion and migration were enhanced. These results suggest that some soluble factors from reactive microglia are capable of enhancing the expression of ICAM-1 on the brain EC. As a consequence, large numbers of tumor-primed T lymphocytes can adhere to EC and migrate across the EC monolayer.     

Involvement of MHC class I molecule and ICAM-1 in the enhancement of adhesion and cytotoxic susceptibility to immune effector cells of tumor cells transfected with the interleukin (IL)-2, IL-4 or IL-6 gene

To investigate the molecular and cellular mechanisms involved in the reduced tumorigenicity and increased immunogenicity of interleukin-2 (IL-2)-, IL-4- or IL-6-gene-transfected B16 melanoma vaccine, we have analyzed the functional and phenotypic properties of these genetically engineered melanoma cells in the present study. The cytokine-gene-transfected B16 melanoma cells showed stronger adhesion to the lymphokine-activated killer (LAK) cells or cytotoxic T lymphocytes (CTL), and higher sensitivity to cytotoxicity of LAK cells or CTL. Using fluorescence-activated cell sorting analysis, we found that both MHC class I and ICAM-1 expression were increased after IL-2, IL-4 or IL-6 gene transfection. The increased level of MHC class I and ICAM-1 expression seems to be responsible for the high sensitivity of these gene-transfected B16 cells to LAK or CTL cytotoxicity because anti-(MHC class I) or anti-ICAM-1 mAb could inhibit the adhesion and cytotoxicity increment simultaneously. The CTL induction was partly inhibited by anti-ICAM-1 mAb and was completely blocked by anti-MHC class I mAb. These results suggested that the decreased tumorigenicity of IL-2-, IL- 4-, and IL-6-gene-transfected B16 melanoma cells may be partly due to the increased sensitivity to effector cell cytotoxicity mediated by increased expression of ICAM-1 or MHC class I molecules on the tumor cell surface after cytokine gene transfection.     

Compartmentalized roles for leukocytic adhesion molecules in lung inflammatory injury

By using the model of acute injury caused by intrapulmonary deposition of IgG immune complexes, blocking mAb to CD11a, CD11b, L-selectin, and intercellular adhesion molecule-1 (ICAM-1) were administered either i.v. or intratracheally (i.t.). The effects of these interventions were assessed according to lung injury, lung content of myeloperoxidase (MPO), TNF-alpha, and cellular content in bronchoalveolar lavage (BAL) fluids, and up-regulation of pulmonary vascular ICAM-1. In animals treated i.v. with Abs to CD11a, L-selectin, or ICAM-1 lung injury was significantly attenuated in parallel with reduced lung content of MPO. Under similar conditions, treatment with anti-CD11b had no effect. However, when the same mAb were administered i.t., anti-CD11a and anti-L-selectin were without protective effects, whereas i.t. administered anti-CD11b and anti-ICAM-1 were each highly protective. The protective effects of anti-CD11b were related to profound reductions in BAL levels of TNF-alpha, pulmonary vascular up-regulation of ICAM-1, and lung content of MPO. The protective effects of i.t.-administered anti-ICAM-1 were not associated with reduced BAL levels of TNF-alpha. Protective effects of mAb were also reflected in reductions of retrievable neutrophils in BAL fluids. mAb to rat CD11b and CD18 but not to rat CD11a suppressed in vitro production of TNF-alpha by immune complex-stimulated rat alveolar macrophages. The mAb did not reduce NO2-/NO3- generation in stimulated macrophages but all mAb (except anti-ICAM-1) reduced O2- responses in macrophages. These data suggest a compartmentalized role for adhesion molecules in lung inflammatory injury after intraalveolar deposition of IgG immune complexes, with CD11a, L-selectin, and ICAM-1 being important in the vascular compartment for neutrophil recruitment, whereas in the alveolar compartment CD11b and ICAM-1 (but not CD11a and L-selectin) seem to play key roles.     

Monocrotaline pyrrole induces apoptosis in pulmonary artery endothelial cells

Eosinophilic myocarditis followed by fibrosis of the cardiac muscle was observed in addition to peripheral blood eosinophilia in CBA/J mice infected with Toxocara canis. The infected mice were used as an experimental model of eosinophilic endomyocarditis associated with hypereosinophilic syndrome. Effects of in vivo treatment with MoAbs to adhesion molecules on eosinophilic myocarditis were examined using this experimental model. Expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells of capillaries in myocardium were increased 1 and 2 weeks after infection. Infiltration of very late antigen (VLA)-4+ and/or CD11a+ cells into the cardiac muscles was also observed 1 and 2 weeks after infection. Infiltration of eosinophils into the heart was significantly suppressed by anti-CD18 MoAb and anti-VLA-4 MoAb, and focal fibrosis of the cardiac muscle was also significantly suppressed by combined administration of anti-CD18 and anti-ICAM-1 MoAbs. These results indicate that adhesion molecules may play important roles in eosinophilic myocarditis, and that blockade of interaction between adhesion molecules and their ligands may help to control it.     

Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation

The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- kappaB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50(-/-) mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50(-/-) mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50(-/-) mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-kappaB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.     

Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells

In bronchial asthma, eosinophils (EOS) adhere to, and migrate across, the lung microvasculature to exert their effector functions in the airways. This study was conducted to determine the effect of cytokines on adhesion molecule expression on human pulmonary microvascular endothelial cells (HPMEC) and the influence of these molecules on EOS adhesion and transmigration in vitro. Unlike ICAM-1 expression (>80% positive cytokine-treated HPMEC by flow cytometry), VCAM-1 expression varied with the cytokine(s) pretreatment; the order of potency was: TNF-alpha + IL-4 (82.2 +/- 4.2% positive cells) > TNF-alpha (41.8 +/- 5.1%) > IL-1beta (20.8 +/- 4.7%). IL-4 alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion to cytokine-treated HPMEC followed the same order as that observed for VCAM-1 expression. Interestingly, EOS migration across cytokine-treated HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to, HPMEC; IL-1beta (21.2 +/- 1.4% migration) > TNF-alpha (12.6 +/- 2.6%) > TNF-alpha + IL-4 (9.1 +/- 2.0%). EOS adhesion was greatest with TNF-alpha + IL-4-treated HPMEC, was dependent on VCAM-1, and inhibited with anti-alpha4 integrin mAb (67.7 +/- 7.5% inhibition, p < 0.0005). In contrast, the highest EOS migration occurred across IL-1beta-treated HPMEC and was inhibited by anti-beta2 integrin mAb (40.4 +/- 2.5% inhibition, p < 0.005). Viable HPMEC were required for EOS migration but not adhesion. Our results suggest that EOS adhesion and transmigration are differentially regulated by VCAM-1 and ICAM-1 expression and the interaction of these adhesion proteins with their respective counterligands, i.e., alpha4 and beta2 integrins on EOS.     

The roles of CD11/CD18 and ICAM-1 in acute Pseudomonas aeruginosa-induced pneumonia in mice

Neutrophil accumulation in response to Pseudomonas aeruginosa in the lungs is mediated through CD11/CD18. This study determined the roles of CD11a, CD11b, and intercellular adhesion molecule (ICAM)-1 in P. aeruginosa-induced pneumonia and compared the function of ICAM-1 using Abs or ICAM-1 mutant mice. Anesthetized BALB/c mice pretreated with either Abs against CD11a, CD11b, ICAM-1, or rat IgG received intratracheal instillation of P. aeruginosa for 4 h. In other studies, ICAM-1 mutant and wild-type mice received either anti-ICAM-1 Ab or rat IgG followed by instillation of P. aeruginosa. The data show that Abs against CD11a, CD11b, and ICAM-1 in BALB/c mice inhibited neutrophil emigration by 79, 81, and 56%, respectively. ICAM-1 mutant mice showed no inhibition of neutrophil emigration compared with wild-type mice. Pretreatment with anti-ICAM-1 Ab inhibited neutrophil emigration in wild-type (129/SvxC57) mice by 67% but had no effect in ICAM-1 mutant mice, suggesting that the Ab was acting specifically through recognition of its Ag. We conclude that CD11a and CD11b are required for neutrophil emigration. The observed function of ICAM-1 varies depending on the method by which it is inhibited. Abs may overestimate function by altering other cellular functions or mutant mice may develop alternative pathways of emigration.     

The effect of substituted laminin A chain-derived peptides on the conformation and activation kinetics of plasminogen

1. The role of the adhesion glycoproteins CD18 and intercellular adhesion molecule-1 (ICAM-1) in inflammatory responses produced during a reversed passive Arthus (RPA) reaction and induced by zymosan and zymosan-activated plasma (ZAP) were studied in rabbit skin. 2. Oedema formation and haemorrhage were quantified by measuring accumulation of 125I-albumin and 111In-labelled red blood cells (111In-RBC) respectively. 3. Monoclonal antibody (mAb) R15.7 (anti-CD18), administered intravenously, abolished accumulation of 125I-albumin and 111In-RBC in dermal RPA reactions and in response to locally injected zymosan and ZAP. 4. When administered intravenously, the mAb RR1/1 (anti-ICAM-1) suppressed 125I-albumin and 111In-RBC accumulation in dermal RPA reactions and at sites treated with zymosan and ZAP. 5. Oedema formation in response to platelet-activating factor (PAF) and bradykinin (BK) either in the presence or absence of prostaglandin E2 (PGE2) were not affected by mAb R15.7 or by mAb RR1/1.1.1. 6. We conclude that oedema formation and haemorrhage associated with RPA reactions and in responses to zymosan and ZAP are completely CD18-dependent, and are mediated, at least in part, via ICAM-1. Responses to the neutrophil-independent oedema forming mediators, PAF and BK are not dependent upon CD18 or ICAM-1.     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.     

Costimulation through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness

The recruitment of eosinophils into the airways after allergen exposure is dependent on interleukin (IL) 5 secreted from antigen-specific CD4+ T cells of the T helper cell (Th) 2 subset. However, while it is established that costimulation through CD28 is required for TCR-mediated activation and IL-2 production, the importance of this mechanism for the induction of a Th2 immune response is less clear. In the present study, we administered the fusion protein CTLA-4 immunoglobulin (Ig) into the lungs before allergen provocation to determine whether CD28/CTLA-4 ligands are required for allergen-induced eosinophil accumulation and the production of Th2 cytokines. Administration of CTLA-4 Ig inhibited the recruitment of eosinophils into the lungs by 75% and suppressed IgE in the bronchoalveolar lavage fluid. CTLA-4 Ig also inhibited the production of IL-4, IL-5, and IL-10 by 70-80% and enhanced interferon-gamma production from CD3-T cell receptor-activated lung Thy1.2+ cells. Allergen exposure upregulated expression of B7-2, but not B7-1, on B cells from the lung within 24 h. Moreover, airway administration of an anti-B7-2 monoclonal antibody (mAb) inhibited eosinophil infiltration, IgE production, and Th2 cytokine secretion comparable in magnitude to that observed with CTLA-4 Ig. Treatment with an anti-B7-1 mAb had a small, but significant effect on eosinophil accumulation, although was less effective in inhibiting Th2 cytokine production. The anti-B7-2, but not anti-B7-1, mAb also inhibited antigen-induced airway hyperresponsiveness in vivo. In all of the parameters assessed, the combination of both the anti-B7-1 and anti-B7-2 mAb was no more effective than anti-B7-2 mAb treatment alone. We propose that strategies aimed at inhibition of CD28 interactions with B7-2 molecules may represent a novel therapeutic target for the treatment of lung mucosal allergic inflammation.     

Engagement of ICAM-3 activates polymorphonuclear leukocytes: aggregation without degranulation or beta 2 integrin recruitment

ICAM-3 is a preferred counterreceptor for the leukocyte alpha(L)beta2 integrin. It activates T cells through outside-in signaling, but polymorphonuclear leukocytes (PMN) are reported to be refractory to ICAM-3 stimulation. We found that engagement of ICAM-3 by a mAb (CAL3.10), which binds in the region where alpha(L)beta2 integrin binds, activates PMN homotypic aggregation and adhesion to surfaces. These functional changes were due to ICAM-3 outside-in signaling because aggregation and adhesion were beta2 integrin-dependent, tyrosine kinase and protein kinase C activities were activated, and there was a reorganization of the cytoskeleton. This reorganization and kinase activity was required for ICAM-3-, but not FMLP-, induced aggregation. This is not an Fc-mediated event as an appropriate anti-ICAM-3 F(ab')2 fragment still induced aggregation. Another anti-ICAM-3 Ab (HP2/19), which activates T cells, did not activate PMN. Strikingly, anti-ICAM-3 did not induce degranulation or cause an increase in surface beta2 integrin expression, so adhesion and aggregation were due solely to the activation of the constitutively expressed beta2 integrins. Aggregation in response to ICAM-3, but not FMLP, was compromised at lower cell densities, showing that beta2 integrin recruitment enhances aggregation under suboptimal conditions. We conclude that engagement of ICAM-3 stimulates PMN as well as T cells, but that the appropriate epitope varies between these two cells. ICAM-3 outside-in signaling reorganizes the cytoskeleton without causing degranulation, induces serine and tyrosine kinase activation, and activates existing surface beta2 integrins to a proadhesive state.