Abstract
The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.
Highlights
The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes
ICAM-1 is usually anchored to the membrane, a soluble ICAM-1 molecule has been identified in serum. sICAM-1 is presented in serum from healthy humans at concentrations between 100 and 450 ng/ml [13] and increased levels of sICAM-1 have been found in serum from patients with cardiovascular and inflammatory diseases as well as during cancer metastasis [14, 15]
We have shown that purified human recombinant ICAM-1 was able to interact with peptides derived from human LFA-1 [34]
Summary
The synthetic first two domains of ICAM-1 alone (D1D2) or linked with human IgG1 Fc region (D1D2Fc) were expressed in Escherichia coli and purified by single step column refolding as described [34]. The ability of the human chimera D1D2Fc to interact with a peptide derived from its natural ligand LFA-1 in mouse was analyzed by ITC on a VP-ITC calorimeter (MicroCal) at 25°C as indicated previously [34]. To check whether the human chimeras of ICAM-1 and human and mouse peptides from LFA-1 were able to recognize their counterpart in mouse cells, a cytotoxicity assay with lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells generated in vivo and EL4 cells as targets as previously described [40] was carried out. CD8 cells were resuspended in RPMI medium 5% decomplemented FBS and preincubated in the presence of several concentrations of human D1D2, D1D2Fc, mouse D1D5Fc, 250 μM of human and mouse LFA-1 derived peptides, human IgG1 15 μg and lovastatin 100 μM for 45 min at 37°C 5% CO2. In both cases, the cells were incubated alone with the inhibitors to evaluate their possible toxicity, which was not significant in any case
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