(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
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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-
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
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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
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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 9095%. 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)-
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
manufacturers 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.
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
(
CD97), which was 84 bp smaller than the amplicon derived from CD97
cDNA (exons 710, 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.
 |
RESULTS
|
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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.
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All cells displayed the strongest expression values consistently for
the CD97 (EGF 1,2,5) isoform, which was responsible for 4065% 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-
. 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 8095 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 35).

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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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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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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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 |
DISCUSSION
|
|---|
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-
, 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.
 |
Note added in proof:
|
|---|
In a recent publication [Lin et al. (2000) Genomics 67,
188200] 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.
 |
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