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
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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
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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.
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or 50 ng/ml tumor necrosis factor (TNF)-
(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
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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
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 CD97 amount necessary
for adjustment of the ratio CD97/CD97cDNA to 1. The lowest amount of
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.
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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.
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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-. 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- 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- (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- (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--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- or 250 U/ml IFN-.
[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).
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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.
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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-, exposition of the cells to IFN- 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.
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Received July 8, 1999; revised March 28, 2000; accepted April 10, 2000.
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ej
í, V., van Lier, R. A. W. (1997) CD97 workshop panel report Kishimoto, T. Goyert, S. Kikutani, H. Mason, D. Miyasaka, M. Moretta, L. Ohno, T. Okumura, K. Shaw, S. Springer, T. A. Sugumura, K. von dem Borne, A. E. G. K. Zola, H. eds. Leucocyte Typing VI: White Cell Differentiation Antigens ,527-529 Garland Publishing New York. This article has been cited by other articles:
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