Development of a cell-free binding assay for rat ICAM-1/LFA-1 interactions using a novel anti-rat LFA-1 monoclonal antibody and comparison with a cell-based assay.

The importance of the interaction between lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in the progression of inflammatory responses in vivo has been demonstrated mainly in rats. The present study was undertaken to develop binding assays suitable for measuring the rat ICAM-1/LFA-1 interaction in vitro. We first examined binding of rat T lymphoma FTL43 cells, which express LFA-1, to immobilized rat ICAM-1. Although FTL43 cells bound avidly to immobilized ICAM-1 and the binding was abolished with anti-LFA-1 monoclonal antibodies (mAbs), the binding was not completely inhibited by most anti-ICAM-1 mAbs. We next purified rat LFA-1 from FTL43 cells and constructed a cell-free binding assay. By using a newly developed anti-rat LFA-1 mAb RL14/9, which does not inhibit ICAM-1/LFA-1 interactions, binding of purified rat LFA-1 to immobilized ICAM-1 was successfully detected, whereas only a low signal to noise ratio was observed when binding of ICAM-1 to immobilized LFA-1 was examined. Moreover, we found that simultaneous addition of purified LFA-1 and biotinylated RL14/9 to ICAM-1-coated wells resulted in more sensitive detection of rat ICAM-1/LFA-1 binding. The binding was completely blocked with both anti-LFA-1 and anti-ICAM-1 mAbs and was much more sensitive to inhibition by the ICAM-1-IgG chimera, as compared with the cell-based assay. These results indicate that the cell-free binding assay provides a rapid and sensitive method for screening rat ICAM-1/LFA-1 antagonists, whose therapeutic effect on inflammatory diseases can further be evaluated in vivo.

Constitutive expression of ICAM-1 in rat microvascular systems analyzed by laser confocal microscopy.

The present study aimed to demonstrate constitutive expression of the intercellular adhesion molecule (ICAM)-1 among arterioles, capillaries, and venules in the mesentery and liver and to examine the interaction between cultured endothelial cells and leukocytes in rats. ICAM-1 expression in the microvessels in vivo was visually demonstrated by laser confocal fluorescence microscopy. A monoclonal antibody against rat ICAM-1 (1A29) was labeled with fluorescein isothiocyanate, and the binding ratio between the fluorescence and immunoglobulin was determined for data calibration. Intravascularly administered fluorescein isothiocyanate-labeled 1A29 was distributed heterogeneously among the hierarchy of microvessels in the mesentery: postcapillary venules were the major portion expressing ICAM-1 constitutively, and the density of 1A29 bound to their endothelium was at least 10 times higher than that in true capillaries and arterioles in the same mesentery. On the other hand, the liver expressed ICAM-1 abundantly in sinusoids to the extent similar to that in central venules. These results suggest that postcapillary venules serve as an active gateway with the readiness to help adhere circulating leukocytes exposed to proinflammatory stimuli in acute inflammation.

Sequence and expression of rat ICAM-1.

We have isolated cDNA clones-coding for rat intercellular adhesion molecule-1 (RICAM-1) from a cDNA library constructed from rat Ax cells stimulated with IL-1 beta using the mouse ICAM-1 cDNA as a hybridization probe. The RICAM-1 sequence shows 79.1% homology with mouse ICAM-1 and 55.6% homology with human ICAM-1 at the nucleic acid level. In order to examine the expression of RICAM-1 on Chinese hamster ovary (CHO) cells, we constructed the vector, pSV-RICAM1-neo, containing the SV40 promoter. Flowcytometric analysis showed that CHO-K1 cells transfected with pSV-RICAM1-neo expressed high amounts of RICAM-1 on their surfaces.

ICAM-1-dependent pathway is critically involved in the pathogenesis of adjuvant arthritis in rats.

Intercellular adhesion molecule 1 (ICAM-1) plays important roles in immune responses. In order to examine whether ICAM-1 is involved in pathogenesis of adjuvant arthritis (AA), we investigated the effect of anti-ICAM-1 mAb, 1A29, on AA in rats. In vivo administration of 1A29 exerted a very strong suppressive effect on the development of arthritis and induced a marked reduction of inflammatory parameters. 1A29 suppressed the Ag-specific proliferative response of lymph node cells from AA rats, suggesting that the mAb blocked the Ag recognition phase. The study using adoptive transfer of AA revealed that 1A29 completely inhibited production of arthritogenic lymphocytes in donors and partially suppressed progression of arthritis in recipients caused by these lymphocytes. These findings indicated that the inhibitory effect of 1A29 on development of arthritis was at least twofold, i.e., 1) interference with cell-cell interaction between APC and T cells, which resulted in abrogation of effector cell generation; and 2) blocking of effector cell migration to inflammatory lesions. These results indicated that ICAM-1-dependent pathway is critically involved in the pathogenesis of AA. The data support the concept that ICAM-1-dependent pathways are important in chronic inflammatory disease.

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.

Thy-1-positive dendritic epidermal cells contain a killer protein perforin.

The killer cell characteristics of Thy-1-positive dendritic epidermal cells (Thy-1+ DEC) were examined. Four Thy-1+ DEC clones which were established from athymic nude mice exhibited spontaneous or lectin-redirectable cytotoxic activity against some murine tumor cell lines in a 4 h 51Cr-release assay. A colorimetric assay for benzyloxycarbonyl-L-lysine-thiobenzyl ester esterase revealed a strong serine esterase activity expressed in all cell clones. In addition, Northern blot analysis using a murine perforin cDNA probe revealed that all four Thy-1+ DEC clones expressed abundant mRNA for perforin, as do most killer T cells. More importantly, immunocytochemical staining with an anti-perforin monoclonal antibody revealed that not only all four Thy-1+ DEC clones but also a part of freshly isolated Thy-1+ DEC from normal and nude mice contained perforin. These results demonstrate that Thy-1+ DEC exhibit typical killer cell characteristics in vitro and in vivo. These data also suggest that Thy-1+ DEC may play a cytotoxic role in protecting the integrity of skin from infection or neoplastic transformation.

Chemotherapeutic efficacy of ofloxacin against experimental pneumonia with Pseudomonas aeruginosa in guinea pigs.

The chemotherapeutic efficacy of ofloxacin against experimental pneumonia was investigated with special reference to its treatment regimen. A pneumonia model was successfully produced by inhalation of a virulent strain of Pseudomonas aeruginosa in guinea pigs immunosuppressed with cortisone acetate. One or 4 days after infection, the animals were treated orally with the fixed daily doses of ofloxacin either once a day or thrice a day for 3 consecutive days. Ofloxacin given thrice a day eliminated the organisms from the lung more efficiently than the equivalent total doses injected once a day in both series of treatment, starting 1 or 4 days after infection. The superiority of the triple dosing in chemotherapeutic efficacy of ofloxacin was found to be attributable at least to the longer retention of its pulmonary levels exceeding the antibiotic concentrations.

CD2 expression in murine B cell lineage.

CD2 expression in murine B cell lineage was examined by flow cytometric and immunoprecipitation studies with anti-murine CD2 mAb and by Northern blot analysis. Cell surface expression of CD2 was demonstrated on all peripheral B cells and cell lines of B lineage. The murine CD2 transcript of 1.3 kb was detected in these B cells. An identical glycoprotein of 55-67 KD was precipitated from the lysates of surface radioiodinated thymocytes, splenic T and B cells, T and B lymphomas, RL male 1 and BCL-1, with anti-murine CD2 mAb. The majority of bone marrow B cells and a half of pre-B cells were found to be positive for CD2 expression. These results indicate that murine CD2 is expressed on B cell lineage at certain differentiation stages.