Elsevier

Human Immunology

Volume 60, Issue 4, April 1999, Pages 323-330
Human Immunology

Original Articles
Differential surface expression of MICA by endothelial cells, fibroblasts, keratinocytes, and monocytes

https://doi.org/10.1016/S0198-8859(98)00128-1Get rights and content

Abstract

MICA is a new, highly divergent and polymorphic HLA-related gene that has a similar intron-exon organization as the HLA class I genes. It functions as a restriction element for intestinal γδT cells and it behaves as a cell stress molecule. It is likely that the polymorphic MICA molecule may be target for specific antibodies and T cells in solid organ grafts or in graft versus host disease (GVHD). Previously, we generated three MICA-specific sera in rabbits, which were used for Western blot, flow cytometry and immunoprecipitation. We demonstrated that MICA is expressed in endothelial cells, keratinocyes and monocytes, but not in CD4+, CD8+ or CD19+ lymphocytes. We also found that MICA is expressed on the cell surface in HeLa cells. In the present work, performing peptide neutralization assays, we further confirmed the specificity of the reactivity of these sera against MICA. Also, by Western blot we demonstrate that freshly isolated human skin-derived fibroblasts express MICA. We also investigated the surface expression of MICA in different, freshly-isolated cells. The results show that endothelial cells and fibroblasts express MICA at the cell surface. Although expressing the 62 kDa MICA band, as detected by Western blots, keratinocytes and monocytes do not seem to express this antigen on the cell membrane. This differential surface expression of MICA by endothelial cells and fibroblasts vs. keratinocytes and monocytes, may indicate that the levels of surface MICA are differentially regulated in different cells. Moreover, the expression of MICA on the surface of endothelial cells makes this polymorphic molecule a possible target during the immune response of graft rejection in organ transplantation.

Introduction

Classical class I human major histocompatibility antigens (HLA-A, -B and -C) are highly polymorphic, 44 kDa membrane-anchored glycoproteins, non-covalently associated with the monomorphic, 12 kDa, β2-microglobulin (β2-m). They are ubiquitously expressed in all nucleated cells of the body, and their main function is to present endogenously-derived peptides of 8 or 9 amino acids, to specific T cells [1]. These T cells express mainly the αβ T cell receptor, and the CD8 co-receptor. The recognition of a foreign peptide (viral, bacterial, or other infectious agent) presented by an HLA class I molecule, together with the engagement of CD8 and other co-receptors, induces a T cell effector function, and in the case of CD8+ T cells, induction of cytotoxicity towards the infected cell [2].

In addition to the classical HLA antigens, there is a group of less polymorphic molecules called non-classical HLA antigens. They are also membrane-anchored glycoproteins, but their pattern of tissue expression is more restricted. Little is known about HLA-F [3], but HLA-E and HLA-G seem to play an important role as recognition molecules for some NK receptors 4, 5. This allows the cells to inhibit the cytotoxic activity of NK cells and seems to be specially important in the maternal-fetal interphase, where the classical HLA antigens are not expressed [6].

More recently, a new family of polymorphic HLA related genes has been described [7]. This family was called MIC (MHC class I related chain), and consists of three pseudogenes (MICC, MICD and MICE) and two expressed genes (MICA and MICB). This gene family is highly divergent from the other HLA genes. The MICA gene maps close to HLA-B and displays an overall homology with the HLA-A, -B or -C genes of less than 40% [7].

Taking into account the structure of the MICA gene, it has been suggested that the encoded polypeptide (383 amino acids in length) has three extracellular domains (α1, α2, and α3), one transmembrane region, and a cytoplasmic tail [7]. It has been speculated that the MICA polypeptide may fold similarly to HLA-A2 [7].

Although the biological function of MICA and MICB still remains unknown, it has been demonstrated that MICA is not up-regulated by IFN-γ [7]. Instead, it behaves as a cell stress antigen, being up-regulated by heat shock [8].

In previous experiments, we retrieved the sequence of one allele of MICA (Genbank accession number L14848 [7]) and synthesized a peptide called MA42-60 (that spans amino acid residues 42-60 of the translated sequence of the gene, and that corresponds to a stretch of the α1 domain), and a peptide called MA140-160 (that spans amino acid residues 140-160 of the translated sequence of the gene, and that corresponds to a stretch of the α2 domain). Using these 2 peptides, we raised 3 sera in rabbits. Serum #620, raised against MA42-60 (α1 domain peptide), and serum #621 raised against MA140-160 (α2 domain peptide), react by Western blot with different cells, detecting a band of about 62 kDa, that presumably corresponds to the denaturated MICA protein [9]. On the other hand, serum #622, also raised by immunization with MA140-160 (α2 domain peptide), immunoprecipitates a band of 62 kDa from metabolically labeled cells, and reacts with HeLa cells by flow cytometry, presumably recognizing the native MICA molecule [9].

Using these immune sera, we have demonstrated that freshly isolated monocytes, keratinocytes, and umbilical vein endothelial cells show, by Western blot, a band of about 62 kDa that corresponds to the MICA protein [9]. This band was not detected in freshly isolated peripheral blood CD4+, CD8+ or CD19+ lymphocytes. Moreover, we also demonstrated that MICA, unlike all other HLA class I antigens, is not associated with β2-microglobulin [9]. Another characteristic that makes MICA different form other HLA class I antigens is that it is not up-regulated by interferon-γ [9]. However, in that study we only analyzed the surface expression of MICA in HeLa cells, and we did not address if there is a differential surface expression of this antigen in cells of different lineages.

More recently, it has been demonstrated that MICA may function as restriction element for a subpopulation of intestinal γδT cells (T cells with δ1 TCR chain). This recognition leads to the lysis by cytotoxicity of the intestinal epithelial cells that express MICA [10].

The aim of the present work was to further confirm the specificity of the reactivity of the anti-MICA rabbit immune sera, to extend the analysis of the expression of MICA in freshly isolated human skin-derived fibroblasts, and to investigate the surface expression of MICA in different, freshly isolated cells.

Section snippets

HeLa cell line

HeLa (ATCC CCL-2, epithelial phenotype) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and was cultured with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) in Dulbecco’s modified Eagle’s medium (DME, Sigma, Saint Louis, MO), supplemented with sodium pyruvate (Sigma), glutamine (Sigma), non-essential amino acids (Sigma), and penicillin/streptomycin (Sigma).

Endothelial cells

Human umbilical vein endothelial cells (HUVECs) were isolated as previously described [9]. Briefly, umbilical

Flow cytometry

Cells were incubated with 10 μl of rabbit serum #622 or the preimmune control for 30 min on ice and washed twice with 1% FCS in PBS and 0.1% sodium azide. Thereafter, 20 μl of fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Cappel, Durham, NC) were added to each tube and incubated for 30 min on ice. After two washes, cells were analyzed in a Coulter EPICS PROFILE II Flow cytometer (Coulter, Miami, FL). Results were expressed as mean fluorescence channels after conversion to a

Results

We previously described the production of the anti-MICA peptide sera #620, #621, and #622 [9]. These sera were used to analyze the pattern of cellular expression of MICA. In order to confirm the specificity of the rabbit sera for MICA, in the present study we investigated the ability of the peptides to neutralize the antibodies by preincubating each serum with the MICA-derived peptides MA42-60 and MA140-160, and analyzing the reactivity of the neutralized serum against its antigen (Fig. 1). As

Discussion

We previously described the production of polyclonal antibodies against MICA by immunizing rabbits with peptides from the translated protein sequence [9]. In order to raise specific polyclonal antibodies, we selected 2 peptides that might correspond to exposed regions of the postulated native protein and located in non-polymorphic regions of the MICA antigen [7]. Thereafter, we searched the nucleotide and protein databases (BLAST search) of the National Center for Biotechnology Information

Acknowledgements

This work was supported in part by NIH Grants RO1HL47145 and UO1AI34621.

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