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(Journal of Leukocyte Biology. 2000;68:561-567.)
© 2000 by Society for Leukocyte Biology

CD97 isoform expression in leukocytes

Wolfram Eichler

Faculty of Biosciences, Pharmaceutics and Psychology, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany

Correspondence and present address: Dr. W. Eichler, University of Leipzig, Interdisciplinary Centre for Clinical Research, Department of Ophthalmology, Liebigstrasse 10-14, D-04103 Leipzig, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note added in proof:...
 REFERENCES
 
Different adhesive capacity in interactions with CD55 has been ascribed to the isoforms of the leukocyte CD97 antigen, CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5). In the study, coexpression of the three CD97 isoforms and predominance of CD97 (EGF 1,2,5) transcripts in leukocytes are demonstrated. The contribution of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) to total CD97 levels varied among most cell types only slightly, although relatively higher mRNA levels of both isoforms were detected in U 937 cells and monocytes. In peripheral blood lymphocytes, CD97 isoforms did not show clear variation after PMA stimulation and were down-regulated equally after CD97 cross-linking. Moreover, the CD97 isoform pattern was not altered in monocytes after interferon-{gamma} stimulation and in synovial T cells from patients with rheumatoid arthritis. CD97 mRNA levels did not necessarily correspond to CD97 surface density. The findings suggest that adhesive activity of CD97 toward CD55 is unlikely to be regulated by differential CD97 isoform expression.

Key Words: leukocytes • CD97 isoforms • EGF-like domains • CD55


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note added in proof:...
 REFERENCES
 
The biological role of the leukocyte surface antigen CD97 is still unknown, although the structural properties of this molecule strongly suggest a functional significance. Thus, expression cloning and sequencing of human [1 ] and murine [2 ] CD97 cDNAs indicated that the CD97 antigen is represented by a serpentine transmembrane protein with an extended extracellular region [1 ]. The extracellular part of CD97 contains a variable number of epidermal growth factor (EGF)-like domains, which give rise to the existence of different CD97 isoforms [3 ]. Similar structural architecture was also described for EMR1 [4 ] and the murine macrophage cell-surface marker F4/80 [5 ]. Based on sequence similarities in their membrane-spanning regions, all of these molecules form a novel class of seven-span (7-TM) proteins (EGF-TM7 family) within the secretin/vasoactive intestinal peptide hormone receptor (SecR) family [6 , 7 ]. Although a conclusive function of these molecules remains to be elucidated, their chimeric structure indicates adhesive and/or signal transducing capability. In line with possible adhesive properties, CD55 (decay-accelerating factor, DAF), a membrane regulatory protein of complement activation, was identified as a cellular ligand for CD97, strongly suggesting that CD97 and CD55 molecules participate in adhesive cellular contacts [8 ].

The human CD97 molecule is expressed in cells and cell lines of different origin with a preference of surface expression to leukocytes [9 ]. However, resting lymphocytes exhibit low levels of cell-surface CD97, but activation of these cells leads to strong CD97 up-regulation [10 ]. A variable number of EGF-like domains in CD97 result from alternative splicing of the CD97 precursor transcript. Thus far, three different CD97 isoforms have been shown to possess three (EGF 1,2,5), four (EGF 1,2,3,5), or five (EGF 1,2,3,4,5) EGF-like domains [3 ]. Although the expression of CD97 isoforms was identified in activated human T cells [3 ], their presence in other cell types has not been investigated. The observation that larger CD97 isoforms, CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5), bind with a significantly lower affinity to CD55 (DAF) raised the question of functional importance of different isoform expression [11 ]. Thus, potentially, leukocytes could regulate the strength of interaction with CD55+ve cells via CD97 isoform expression. Because the expression of these isoforms in different cell types has not been compared with each other, the study described in this investigation was undertaken. It encompasses investigations of isoform pattern, mRNA levels, and cell-surface density of CD97 in leukocytes of different lineages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Note added in proof:...
 REFERENCES
 
Preparation, culture, and in vitro stimulation of cells
The human cell lines used in this study (K 562, Daudi, CEM, Jurkat, U 937, HL-60) were obtained from American Type Culture Collection (ATCC; Rockville, MD) and routinely cultured at 2 x 106 cells/ml in complete RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Peripheral blood mononuclear cells (PBMC) were separated from blood of healthy donors by standard density gradient centrifugation (density=1.077). Fractions of lymphocytes and monocytes were obtained by counter-flow elutriation. Purity of these cells was 90–95%. Peripheral blood lymphoctyes (PBL) used to study activation-dependent CD97 antigen expression were stimulated with 5 ng/ml phorbol 12-myristate 13-acetate (PMA; Calbiochem, Bad Soden, FRG). Stimulation of PBL with cross-linked monoclonal antibody (mAb) CD97 mAb BL-Ac/F2 was performed in flat-bottom plates (Greiner, Nuertingen, FRG), precoated with 50 µg/ml sheep anti-mouse immunoglobulin (Ig; Roche Molecular Biochem, Mannheim, FRG). PBL were added to the washed plates and incubated at 37°C for 8 h. Monocytes were stimulated with recombinant 250 U/ml interferon (IFN)-{gamma} or 50 ng/ml tumor necrosis factor (TNF)-{alpha} (R&D Systems, Minneapolis, MN) for 2 days. Synovial fluid was obtained from two patients (one male, age 63 years; one female, age 74 years) with rheumatoid arthritis, conforming to the American College of Rheumatology criteria. The samples were diluted 10-fold in phosphate-buffered saline (PBS) and centrifuged at 400 g for 15 min. Cells were further separated by density gradient centrifugation. T cells of peripheral blood samples and synovial fluid were purified by immunomagnetic separation on ice using incubation for 20 min with CD3-coated Dynabeads (Dynal, Hamburg, FRG).

RNA preparation and polymerase chain reaction (PCR) amplification of CD97 mRNA
Total RNA of cells was prepared using a commercially purchased RNA isolation kit (InViTek, Berlin, FRG), according to the manufacturer’s specifications. The resulting RNA was precipitated using isopropanol, dried, and dissolved in H2O. Contaminating genomic DNA was eliminated with 1 u DNase I (Life Technologies, Eggenstein, FRG). PCR amplification was performed using as template single-stranded cDNA, obtained by reverse transcription of total RNA preparation. The cDNA was synthesized from 1 µg in a 20-µl reaction using 200 u of Superscript II reverse transcriptase (dT)15 (Life Technologies), 500 µM each of nucleotides, and 0.5 µg of oligo(dT)15 (InViTek). PCR was performed within the exponential amplification range using a 20-µl vol with 0.5 u of InViTAQ DNA polymerase (InViTek), 1 µl of single-stranded cDNA, 100 µM deoxynucleotide triphosphates (dNTPs), 125 nM each of the CD97-specific primers (indicated in Table 1 ) in 50 mM Tris-HCl, pH 8.8, 16 mM (NH4)2SO4, 2.5 mM MgCl2, and 0.01% Triton X-100. PCR products were separated by electrophoresis on a 1.8% agarose gel.


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  		Table 1. Human CD97 and G3PDH Oligonucleotides

 
Quantitation of PCR products
Relative CD97 mRNA levels were analyzed by reverse transcription followed by PCR (RT-PCR). The primers used throughout this study are indicated in Table 1 . To analyze total CD97 mRNA, cDNA samples were adjusted to equal G3PDH inputs by PCR in the presence of a competitor (kindly provided by Dr. P. Ruschpler, Institute of Pathology, University of Leipzig, Germany). This competing DNA was 283 bp longer than the amplified fragments (566 bp) derived from G3PDH cDNA samples. An internal standard for a competitive CD97 PCR was constructed as described previously [12 ]. Briefly, a CD97 DNA fragment ({triangleup}CD97), which was 84 bp smaller than the amplicon derived from CD97 cDNA (exons 7–10, 331 bp), was generated by PCR and cloned into the pCR-Script plasmid (Stratagene, Heidelberg, FRG). Known amounts of {triangleup}CD97 were coamplified with unknown amounts of sample CD97 cDNA, competing by the same set of primers. Relative sample CD97 cDNA levels were expressed in arbitrary units (AU) of each {triangleup}CD97 amount necessary for adjustment of the ratio {triangleup}CD97/CD97cDNA to 1. The lowest amount of {triangleup}CD97, which yielded a visible amplification product in agarose gels using ethidiumbromide staining, was defined as 1 AU. PCR of target-derived and competitive fragments occurred with virtually identical efficiency and was performed within the exponential amplification range. Ethidiumbromide-stained agarose gels were scanned using a CMD camera of a GelPrint 2000i Station from BioPhotonics Corp. (Ann Arbor, MI) and analyzed with the Sigmagel Software (Jandel Corp., San Rafael, CA).

Immunofluorescence analysis of CD97 antigen expression
The murine mAb BL-Ac/F2 (IgG1), which is directed to CD97 EGF-like domain 1 and was characterized further at the VIth International Workshop on Leukocyte Differentiation Antigens [13 ], was used in this study. Cell-surface expression of CD97 antigen was analyzed by staining aliquotes of 2 x 105 cells with BL-Ac/F2 followed by incubation with FITC-conjugated goat anti-mouse IgG (Sigma, Deisenhofen, FRG). Samples were analyzed on a flow cytometer (FACScan, Becton Dickinson, San Jose, CA) using LYSYS version 1.1 software. Gates were set to discriminate between different cell populations and to exclude nonviable cells, and histograms were recorded to determine percentage and mean fluorescence intensity (MFI) of labeled cells defined by scatter gates.

Immunoprecipitation
Surface labeling of cells was performed by a modification of a previously described method [14 ] using D-biotin-N-hydroxysuccinimide ester (Boehringer Mannheim, FRG). Cell lysates were prepared on ice by detergent lysis (5x107 cells/ml) in a buffer containing 50 mM Tris-HCl, 0.15 M NaCl, pH 8, 2% Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM ethylene diaminetetraacetate (EDTA), and precleared twice using goat anti-mouse IgG-coated protein A Sepharose and mouse IgG-coupled Sepharose beads. For immunospecific isolation [15 ] of the CD97 antigen, cell extracts were incubated overnight at 4°C with mAb BL-Ac/F2. The absorbed antigens were eluted and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [16 ]. Following electrophoresis, proteins were transferred to nitrocellulose, which was blocked afterward for 2 h at 37°C in 5% powdered nonfat dried milk and probed with streptavidin/alkaline phosphatase (Boehringer Mannheim), followed by 0.5 mg/ml nitrobluetetrazolium and 0.25 mg/ml 5-bromo-4-chlor-3-indolyl phosphate (Sigma). Enzymatic digestion of the isolated CD97 antigen was performed by incubation with 0.5 U endoglycosidase-F (Boehringer Mannheim) in 20 mM phosphate buffer, 50 mM EDTA, pH 6.1, 1% NP-40, 1 mM PMSF, 1% 2-mercaptoethanol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Note added in proof:...
 REFERENCES
 
CD97 isoform pattern in leukocytes
To analyze CD97 isoform expression in leukocytes, CD97 mRNA from PBL, peripheral blood monocytes, and cell lines representative of erythroid, lymphoid, and myeloid cell lineages were analyzed. RT-PCR experiments were performed with primers that flank the exons encoding for the EGF-like domains (Fig. 1 ). The CD97 (EGF 1,2,3,4,5) form (CD97 mRNA with exons 5 and 6) generated a 857-bp PCR fragment; the CD97 (EGF 1,2,3,5) form (mRNA without exon 6) yielded a 710-bp band, and the CD97 (EGF 1,2,5) isoform (exon 5-ve, exon 6-ve) gave rise to a 578-bp band. The results indicated that the isoforms CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5) are expressed in all cells investigated. It was also observed that the presence of exons 5 and/or 6 encoding for EGF-like domains 3 and 4, respectively, is linked in most cells to decreasing levels of the corresponding CD97 isoforms (Fig. 2A ).



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  		Figure 1. Schematic representation of the CD97 structure and position of primers used for amplification of CD97 isoforms. Only the 5' terminal part of mature CD97 mRNA is shown. This part contains the exons that encode for the signal peptide (SP) and EGF-like sequence repeats. The scheme neglects that the start of EGF-like domain 1, as defined in ref. 1 does not match exactly the beginning of exon 3. The EGF-like domains 3 and 4, which are lacking in the CD97 (EGF1,2,3,5) and CD97 (EGF1,2,5) isoforms, respectively, are indicated by shaded boxes. The position of primers (F=forward; R=reverse) is indicated by solid circles.

 


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  		Figure 2. Expression of CD97 isoforms in various human hematopoietic cells and cell lines. (A) Agarose gel showing separation of the CD97 isoforms. (B) Proportions of mRNA encoding for CD97 (EGF1,2,3,5) and CD97 (EGF1,2,3,4,5) within total CD97 mRNA of the indicated cells and cell lines. (C) Relative expression of isoforms CD97 (EGF1,2,3,4,5) and CD97 (EGF1,2,3,5). Pattern of CD97 isoforms was analyzed by RT-PCR, which was performed on 1 µg total RNA with the primers binding to exons 2 and 9 of the human CD97 gene as indicated in Table 1 and Figure 1 . Contrary to B and C, the results shown in A were obtained after RT-PCR analysis outside the exponential amplification range (40 cycles) to ensure clear presentation of the three isoforms in all samples. Agarose gels were ethidiumbromide-stained, scanned using a CMD camera, and analyzed using commercially available software. Concentrations of CD97 mRNA were expressed as proportions of integration values of bands corrected by factors that took into account dependence on different DNA lengths of ethidiumbromide fluorescence. Data are representative of three or more replicate experiments and are given as mean ± standard deviation.

 
All cells displayed the strongest expression values consistently for the CD97 (EGF 1,2,5) isoform, which was responsible for 40–65% of total CD97 mRNA dependent on the cell type investigated. Thus, transcripts lacking sequences from CD97 exons 5 or 6 appear to be generated preferentially in leukocytes. Furthermore, the relative proportions of the two larger isoforms were relatively constant among the cells. Evaluating their contribution to the total CD97 fraction and mutual levels revealed that relative expression of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) varied most between the monocytic cells and the other cell types. First, higher expression of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) was characteristic for U 937 cells. Notably, U 937 cells expressed larger isoforms at a level that exceeds that of CD97 (EGF 1,2,5) (Fig. 2B) . Second, quantitation of CD97 (EGF 1,2,3,4,5) revealed that U 937 cells and monocytes express this isoform stronger, at about 75% of the CD97 (EGF 1,2,3,5) level (Fig. 2C) .

CD97 isoform expression during cellular activation and inflammatory reactions
To determine whether cellular activation leads to variations in CD97 isoform expression, PBL were activated with PMA, and peripheral blood monocytes were stimulated with IFN-{gamma}. The phorbol-ester PMA stimulates T lymphocytes by activating protein kinase C, which is a key event associated with the T-cell antigen receptor/CD3 stimulation. IFN-{gamma} was selected, because this cytokine is able to regulate the expression of cell-surface proteins of the monocytic lineage [17 ]. When CD97 isoform expression in PMA-stimulated PBL was detected by RT-PCR, three distinct amplification products indicated the expression of the CD97 isoforms, CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5). Expression of single isoforms was apparently not influenced during two days of PMA treatment (Fig. 3A ). Similar results were obtained by using phytohemagglutinin-activated PBL (unpublished results). The findings were confirmed by immunoprecipitation of the CD97 protein from lysates of PMA-stimulated and cell-surface-labeled PBL. Following Endo-F digestion of the isolated 80–95 kDa CD97 antigen, three bands of Mr 58, 64, and 71 kDa were visible (Fig. 3B) . These bands, which are consistent with the predicted molecular weights of the processed CD97 isoforms and with previously characterized CD97 isoforms from transfectants [1 , 3 ], suggest that the CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5) proteins are expressed on the lymphocyte cell surface. After activation of monocytes with IFN-{gamma} (Fig. 3C) , a slight increase of the CD97 (EGF 1,2,3,4,5) isoform was observed. The CD97 (EGF 1,2,3,4,5)/(EGF 1,2,3,5) ratio (0.83±0.08) in these cells was slightly higher than in cells cultivated with medium alone (0.75±0.08) or TNF-{alpha} (0.74±0.05), but this effect was not significant (p=0.07). This result suggests that the CD97 isoform pattern in monocytic cells is relatively constant. However, it cannot be excluded that the IFN-{gamma}-induced signal transduction pathway may influence the CD97 isoform expression under other conditions of cellular activation. The purpose of further experiments was to down-regulate CD97 mRNA levels and to examine whether this affects the expression of CD97 isoforms. Therefore, cell-surface CD97 of PBL was cross-linked by the mAb BL-Ac/F2, a treatment that resulted in a decrease of CD97 mRNA. All three CD97 isoforms appeared to be affected equally by this down-regulation (Fig. 3D) . Previously published results [3 , 18 ] indicated a potential role of CD97 expression in inflammatory processes. Therefore, CD97 isoforms were also analyzed in synovial T lymphocytes from patients with rheumatoid arthritis, known to be in an activated state [19 ]. Figure 3E demonstrates that the relative expression of isoforms in synovial T cells (lanes 1 and 2) did not show clear differences from the CD97 isoform pattern of normal peripheral blood T cells (lanes 3–5).



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  		Figure 3. Expression pattern of CD97 isoforms during stimulation of PBMC, after down-regulation of CD97 mRNA in PBL, and in T lymphocytes derived from synovial fluid of patients with rheumatoid arthritis. CD97 mRNA was analyzed by RT-PCR using the CD97-specific primers as in Figure 1 . The position of molecular standards is shown at the margin of each panel. (A) Detection of CD97 isoforms in PBL stimulated with PMA (5 ng/ml) for the indicated periods of time. (B) Immunoprecipitation of the CD97 antigen from PMA-stimulated PBL. At the indicated time points, immunoprecipitates were prepared from NP-40 lysates of surface-labeled cells using the mAb BL-Ac/F2. The isolated CD97 antigen was incubated without (-) or with (+) endoglycosidase F and separated by SDS-PAGE. (C) Detection of CD97 isoforms in monocytes, which were cultured for two days in medium alone, with 50 ng/ml TNF-{alpha} or 250 U/ml IFN-{gamma}. [See text for CD97 (EGF 1,2,3,4,5)/(EGF 1,2,3,5) ratios (n=3).] (D) Down-regulation of CD97 mRNA in PBL. Cells were cultivated for 8 h with cross-linked irrelevant IgG1 mAb (control) or mAb BL-Ac/F2. The cDNA in these samples was adjusted to equal G3PDH inputs and amplified by PCR as indicated in Materials and Methods. (E) CD97 isoforms in synovial T cells obtained from two patients with rheumatoid arthritis in comparison with T cells from normal peripheral blood samples. The CD97 (EGF 1,2,3,4,5)/(EGF 1,2,3,5) ratios in synovial T cells were 0.45 (lane 1) and 0.52 (lane 2), and in normal peripheral blood T cells, 0.32 (lane 3), 0.51 (lane 4), and 0.56 (lane 5).

 
Relationship between CD97 mRNA and surface expression in leukocytes
To obtain more insight in regulatory mechanisms that control a putative CD97-mediated adhesive activity, it was important to know whether mRNA expression and cell-surface density of CD97 are correlated. Therefore, a comparison of relative CD97 mRNA levels with CD97 surface expression was performed using PBL, peripheral blood monocytes, and the cell lines, U 937, HL-60, K 562, CEM, Jurkat, Daudi, and Raji (Table 2 ). The results of semiquantitative RT-PCR analysis are shown in Figure 4 . High CD97 mRNA levels were detected in myeloid (HL-60) or monocytic cells, which were paralleled by strong CD97 surface expression. In contrast, in spite of high CD97 mRNA levels, PBL expressed CD97 only at low density on the cell surface. In general, CD97 surface expression on PBL was comparable to that of the lymphoid cell lines, CEM, Daudi, and Raji. Only a fraction of these cells displayed surface CD97 at a low level, which was consistent with their low CD97 mRNA levels. Surface CD97 staining of Jurkat cells was weak and of even lower magnitude in comparison with the other lymphoid cells. Thus, higher CD97 mRNA expression in PBL and Jurkat T cells does not indicate equally higher CD97 surface density. In these cells, CD97 surface expression is likely to be regulated also independently of the mRNA level, for example, by mechanisms that control the redistribution and/or targeting of the CD97 protein. The results suggest that the surface expression and mRNA levels of CD97 are not necessarily correlated. Moreover, transcriptional regulation of isoform proportions and/or regulation of mRNA stability of CD97 might be mechanisms that do not sufficiently determine a functional CD97 expression.


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  		Table 2. Comparison of CD97 mRNA with Corresponding Cell-Surface Expression in Different Cells and Cell Linesa

 


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  		Figure 4. Representative semiquantitative RT-PCR analysis of CD97 mRNA expression in various cells and cell lines. Serial dilutions of an internal CD97 competitor DNA (indicated above the panels in fg) were added to unknown amounts of sample cDNA. The 331-bp (sample cDNA) and 247-bp (CD97 competitor) PCR products were analyzed by agarose gel electrophoresis and by measuring the intensity of ethidiumbromide fluorescence as indicated in Materials and Methods.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Note added in proof:...
 REFERENCES
 
In this study, expression characteristics of the three CD97 isoforms, CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5), were investigated. Differential splicing of transcripts leading to the generation of isoforms with a variable number of EGF-like domains is a common attribute of the members of the EGF-TM7 family, CD97, EMR-1, and F4/80 [3 , 5 , 20 ]. EGF-like domains, which are similarly arranged as in EGF-TM7 members, can also be found in thrombomodulin/CD141, the low-density lipoprotein receptor, and the extracellular matrix proteins, fibrillin, fibulin, and nidogen/entactin. These molecules are characterized by tandemly arranged EGF-like sequences, the interdomain linkage of which is stabilized by hydrophobic interactions between conserved amino acid residues [21 ]. Additional presence of EGF-like domains may influence the overall rod-like conformation of one given Ca2+-binding EGF domain pair or modulate affinity for Ca2+ of one Ca2+-binding domain [22 ]. Therefore, variable expression of EGF-like domains in EGF-TM7 proteins might have consequences for interactions with putative ligands. Indeed, the additional expression of EGF-like domains 3 and/or 4 in CD97 was linked to reduced binding capacity of CD97-transfected COS-7 cells to CD55 (DAF), which has been identified as a cellular ligand for CD97 [8 ]. Thus the questions raised are (1) whether certain leukocytes express some isoform(s) preferentially, which could give rise to particular CD97-mediated adhesive properties, and (2) whether lineage-specific or activation-dependent differences exist in terms of isoform expression patterns.

The single CD97 isoforms are apparently not expressed in a lineage-specific manner; the triplet of bands after PCR amplification indicates rather a coexpression of the CD97 isoforms. The CD97 (EGF 1,2,5) isoform, which is characterized by a strong adhesive capacity toward CD55 [11 ], was expressed predominantly in different cells and cell lines. No splice variants with less than three EGF-like domains were observed, confirming earlier findings that indicated that the structural and functional integrity of CD97 requires a minimal sequence of three EGF-like domains [11 ]. The contribution of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) to total CD97 levels varied among nearly all cells and cell lines and during cellular activation only slightly. Thus, activation of PBL by PMA was apparently not accompanied by preferential expression of one or both larger isoform(s), at least during the time interval (2 days) investigated. The study of synovial T cells derived from patients with rheumatoid arthritis, which may express early (CD69), late [major histocompatibility complex (MHC) Class II, VLA-4], and very late (VLA-1) activation markers [19 ], suggests that inflammatory processes are not linked with changes of the CD97 isoform pattern. Additionally, down-regulation of CD97 mRNA in PBL was initiated by ligation of the CD97 protein and found to be related to all isoforms. Thus, all available data indicate that proportions of CD97 isoform transcripts are relatively constant in lymphocytes. This is not in accordance with a possible interrelationship between the regulation of the expression of single isoforms and CD97-mediated cellular processes.

In comparison with monocytes cultured in medium alone or in the presence of TNF-{alpha}, exposition of the cells to IFN-{gamma} resulted in only a slightly altered CD97 (EGF 1,2,3,4,5)/(EGF 1,2,3,5) ratio. Although this effect was not significant, it is possible that cytokines influence the balance of CD97 isoforms in monocytes under conditions that are still to be defined. The function of CD97 in monocytic cells and participation of CD97 in adhesive interactions have not been elucidated. However, the lack of a marked shift of the CD97 (EGF 1,2,3,4,5)/(EGF 1,2,3,5) ratio after cytokine exposition is rather consistent with more or less stable CD97 isoform proportions.

Regulatory mechanisms leading to differential CD97 isoform expression are expected to act at the mRNA level. Stronger mRNA expression of distinct isoforms could contribute to an increased CD97 surface density, and thus, it is relevant to possible adhesive interactions. Additionally, generation of high CD97 (EGF 1,2,5) mRNA levels could indicate adequate expression of this isoform on the cell surface and preference for CD97-mediated adhesive contacts. The comparison of the cell-surface expression levels of different cell types suggested that CD97 is functionally significant in monocytic and myeloid cells. However, U 937 cells express CD97 strongly in association with a higher level of the CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) isoforms. Compared with other cell types, U 937 cells and monocytes exhibit an enhanced expression of CD97 (EGF 1,2,3,4,5) in proportion to CD97 (EGF 1,2,3,5) also. Because the larger isoform(s) bind with a significantly lower activity to CD55 (DAF) [11 ], their overrepresentation in monocytic cells might not favor adhesive contacts. Further results indicated that even the relatively high levels of CD97 mRNA in PBL and Jurkat cells did not determine stronger CD97 cell-surface expression. With regard to adhesive contacts of lymphoid cells, even the predominant CD97 (EGF 1,2,5) isoform may be insufficiently expressed on the cell surface; e.g., the preferential expression of CD97 (EGF 1,2,5) is insignificant to CD97 (EGF 1,2,5)-based interactions with CD55 (DAF). A possible explanation for regulation of CD97 cell-surface expression in PBL provided previous experiments that demonstrated that these cells possess intracellular CD97 protein. Its redistribution onto the cell surface contributes to rapid CD97 up-regulation following cellular activation [9 ]. In the context stated above, it must be noted that U 937, HL-60, and lymphoid cells failed to adhere to CD55+ve erythrocytes (unpublished results); this adhesion is a characteristic property of CD97 (EGF 1,2,5)+ve transfectants [8 ]. Thus, regardless of the prominent CD97 (EGF 1,2,5) isoform expression in all leukocytes and the strong CD97 cell-surface expression of monocytic cells, there is no evidence that different proportions of single CD97 isoforms at the RNA level indicate a different adhesive capacity of CD97 on the cell surface. Rather than differential regulation of mRNA levels of CD97 isoforms, additional mechanisms, which determine CD97 protein expression and distribution and/or accessibility of single isoforms to the ligand, are likely to be important. The apparent dependence on such processes in conjunction with the relative invariability of the CD97 isoform ratio in leukocytes suggests that the adhesive activity of CD97 is not predetermined by the proportion of isoforms.


   Note added in proof:
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note added in proof:...
REFERENCES
 
In a recent publication [Lin et al. (2000) Genomics 67, 188–200] the existence of a CD97-like molecule, EMR2, is described. EMR2 shares with CD97 the almost identical EGF-like domains and is also recognized by the CD97 mAb BL-Ac/F2.


   ACKNOWLEDGEMENTS
 
The technical assistance of S. Petter is greatly appreciated. The author is grateful to K. Droessler for his support, to M. Pfister for helpful discussion, and to G. Baumbach and S. Hauschildt for supplying elutriated cells.

Received July 8, 1999; revised March 28, 2000; accepted April 10, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note added in proof:...
 REFERENCES
 

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