(Received for publication, June 12, 1995)
From the
The human activation antigen CD69 is a member of the C-type
animal lectin superfamily that functions as a signal-transmitting
receptor. Although the expression of CD69 can be induced in vitro on cells of most hematopoietic lineages with a wide variety of
stimuli, in vivo it is mainly expressed by T-lymphocytes
located in the inflammatory infiltrates of several human diseases. To
elucidate the mechanisms that regulate the constitutive and inducible
expression of CD69 by leukocytes, we isolated the promoter region of
the CD69 gene and carried out its functional characterization. Sequence
analysis of the 5`-flanking region of the CD69 gene revealed the
presence of a potential TATA element 30 base pairs upstream of the
major transcription initiation site and several putative binding
sequences for inducible transcription factors (NF-B, Egr-1, AP-1),
which might mediate the inducible expression of this gene. Transient
expression of CD69 promoter-based reporter gene constructs in K562
cells indicated that the proximal promoter region spanning positions
-78 to +16 contained the cis-acting sequences
necessary for basal and phorbol 12-myristate 13-acetate-inducible
transcription of the CD69 gene. Removal of the upstream sequences
located between positions -78 and -38 resulted in decreased
promoter strength and abolished the response to phorbol 12-myristate
13-acetate. We also found that tumor necrosis factor-
(TNF-
)
is capable of inducing the surface expression of the CD69 molecule as
well as the promoter activity of fusion plasmids that contain
5`-flanking sequences of the CD69 gene, suggesting that this cytokine
may regulate in vivo the expression of CD69. In addition,
cotransfection experiments demonstrated that the CD69 gene promoter can
be activated by the NF-
B/Rel family members c-Rel and RelA. The
deletion of the sequence spanning positions -255 to -170
abolished both the response to TNF-
and the transactivation by
NF-
B. These results indicate that the NF-
B-binding site
located at position -223 is necessary for the TNF-
-induced
expression of the CD69 gene. Mobility shift assays showed that the two
NF-
B motifs located in the proximal promoter region (positions
-223 and -160) bind various NF-
B-related complexes,
including the heterodimers p50/RelA and p50/c-Rel and homodimers of p50
(KBF-1) and RelA. Our findings help to explain the regulated synthesis
of CD69 in vivo and suggest that TNF-
has a key role in
the expression of this molecule at sites of chronic inflammation.
The human activation inducer molecule (AIM/CD69) is a
phosphorylated disulfide-linked 27/33-kDa transmembrane homodimeric
glycoprotein (Sánchez-Mateos and
Sánchez-Madrid, 1991). The CD69 molecule is
rapidly expressed on the surface of T-lymphocytes upon in vitro activation with a wide variety of agents, including anti-CD3/T
cell receptor and anti-CD2 mAbs, ()activators of protein
kinase C, and phytohemagglutinin (Cebrián et
al., 1988; Hara et al., 1986). Similarly, the expression
of CD69 is inducible on the surface of NK cells, B-lymphocytes, and
eosinophils (Hartnell et al., 1993; Lanier et al.,
1988). In contrast, CD69 is constitutively expressed in vivo on platelets and on a small percentage of resident T- and B-cells
of different lymphoid tissues (Sánchez-Mateos et al., 1989; Testi et al., 1990). Although a
physiologic ligand for CD69 has not been identified so far, experiments
with specific mAbs indicate that this antigen functions as a
signal-transmitting receptor. Signals triggered by CD69 mAbs in
T-lymphocytes include increase in intracellular calcium concentration
and result in the synthesis of different cytokines and their receptors,
enhancement of the expression of c-myc and c-fos protooncogenes, and cell proliferation
(Cebrián et al., 1988; Nakamura et
al., 1989; Santis et al., 1992; Testi et al.,
1989; Tugores et al., 1992). In NK cells and platelets, CD69
also acts as a triggering molecule, being involved in the redirected
target cell lysis by interleukin-2-activated NK cells (Moretta et
al., 1991) and in the induction of platelet aggregation,
Ca
influx, and hydrolysis of arachidonic acid (Testi et al., 1990).
The molecular cloning of cDNAs encoding
human and mouse CD69 revealed that this antigen is a member of the
Cadependent (C-type) animal lectin superfamily of
type II transmembrane receptors (Hamann et al., 1993;
López-Cabrera et al., 1993; Ziegler et al., 1993). This superfamily includes the human NKG2, rat
and mouse NKR-P1, and mouse Ly-49 families of NK cell-specific antigens
as well as the low avidity IgE receptor (CD23), the Kupffer cell
receptor, and the hepatic asialoglycoprotein receptor (Drickamer, 1993;
Yokoyama, 1993). Characterization of the structure of genes encoding
human and mouse CD69 further evidenced that CD69 is evolutionarily
related to these C-type lectin receptors
(Santís et al., 1994; Ziegler et al., 1994). In addition, the CD69 gene is clustered with
other C-type lectin-encoding genes within a genetic region named the NK
cell complex (Drickamer, 1993; López-Cabrera et al., 1993; Yokoyama, 1993; Ziegler et al., 1994).
The CD69 antigen is undetectable on peripheral blood lymphocytes;
however, it is expressed at high levels by the majority of T-cells in
the inflammatory cell infiltrates of several human diseases such as
rheumatoid arthritis and chronic viral hepatitis
(García-Monzón et al., 1990; Laffón et al.,
1991), suggesting that inflammatory cytokines may be involved in CD69
expression. In this context, it has been reported that a large amount
of TNF- is produced by human hepatocytes in chronic viral
hepatitis (González-Amaro et al., 1994).
Thus, TNF-
and other cytokines may play a key regulatory role in
the inducible expression of the CD69 antigen in vivo.
Northern blot analysis demonstrated that the constitutive and
inducible expression of the CD69 molecule is regulated at the
transcriptional level (Hamann et al., 1993,
López-Cabrera et al., 1993; Ziegler et al., 1993, 1994). To determine the molecular basis for the
pattern of CD69 expression, we have isolated the 5`-region of the CD69
gene and analyzed its inducible promoter activity. We describe herein
that the upstream sequence of the CD69 gene functions as a
PMA-inducible promoter element. In addition, we report that TNF-
is capable of inducing both the surface expression of the CD69 antigen
and the promoter activity of the 5`-region of the CD69 gene. The
presence of NF-
B motifs within the proximal promoter region of the
CD69 gene may account for the TNF-
-inducible promoter activity.
Restriction analysis of the clones indicated that they possessed the same 3-kbp EcoRI insert, which was subcloned into the pBluescript plasmid (Stratagene, La Jolla, CA). DNA sequencing was performed by the dideoxy termination method either by subcloning restriction fragments into pBluescript vectors or by direct oligonucleotide-primed DNA sequencing with internal primers.
K562 erythroleukemic cells were
transiently transfected with 4 µg of each recombinant plasmid using
10 µg of Lipofectin reagent (Life Technologies, Inc.) according to
the manufacturer's recommendations. To normalize transfection
efficiency, 1 µg of pCMV-gal (CLONTECH, Palo Alto, CA), which
contains the cytomegalovirus promoter ligated to the
-galactosidase gene, was included in each transfection. After 48 h
of transfection, luciferase activity was determined in cell extracts
containing identical
-galactosidase activity according to the
instructions of the luciferase assay kit (Promega). Light emission was
measured in a Lumat LB9501 luminometer (Berthold, Wildbad, Germany),
and the results are expressed as relative light units. To analyze the
effect of PMA and TNF-
on the CD69 promoter activity, half of the
transfected cells were treated for 12-16 h with either 20 ng/ml
PMA or 50 ng/ml human recombinant TNF-
(3.2
10
units/mg; Wichem, Vienna). As positive controls for PMA and
TNF-
stimulation, we used the plasmids pGL2P (Promega), in which
the luciferase gene is under the control of the SV40 early promoter,
and pKBF-Luc (a gift from Dr. A. Israël, Institut
Pasteur, Paris), which contains a trimer of the NF-
B motif of the
H-2K
gene (Yano et al., 1987) upstream of the
herpes simplex virus thymidine kinase gene promoter.
To obtain K562
stable transfectants, cells were cotransfected by electroporation with
50 µg of pAIM1.4-Luc and 10 µg of pSV2-neo as described
previously (Nueda et al., 1993), selected in the presence of
G418 (1 mg/ml), and cloned by limiting dilution. To analyze the
transactivation of the CD69 promoter by NF-B/Rel family members,
K562 cells were cotransfected, in the presence of 20 µg of
Lipofectin, with 5 µg of CD69 promoter-derived plasmids and
5-10 µg of the expression vector pRc/CMV-p50, pRc/CMV-c-Rel,
or pRc/CMV-RelA (kindly provided by Dr. A. Israël),
which contained the full-length cDNA encoding each protein, into the
pRc/CMV plasmid (Invitrogen, San Diego, CA) (Le Bail et al.,
1993).
Binding reactions for gel retardation assays
were performed at 0 °C in a volume of 20 µl containing 10
mM Hepes, pH 7.6, 10% glycerol, 50 mM KCl 6 mM MgCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 5 µg of poly(dI-dC), 0.5 ng of 3`-end labeled
probe, and 2 µg of nuclear extract. After the binding reaction, the
mixtures were electrophoretically separated on 4-5% nondenaturing
polyacrylamide gels. When indicated, 0.5 µl of rabbit anti-p50
(Kieran et al., 1990), anti-RelA, anti-c-Rel, or anti-p52
specific polyclonal antibodies were added to the corresponding binding
reaction prior to the addition of the radiolabeled probe. These
antisera were kindly provided by Drs. A. Israël and
N. R. Rice. For competition, a 50-fold molar excess of unlabeled
oligonucleotide was added to the binding reaction prior to the addition
of the probe. The sequences of the oligonucleotide probes (and their
complementaries) used in this study were as follows: CD69-
B-1,
5`-GATCAGACAACAGGGAAAACCCATACTTC-3` (nucleotides -170 to
-144); and CD69-
B-2, 5`-GATCAGAGTCTGGGAAAATCCCACTTTCC-3`
(nucleotides -232 to -206). The synthetic oligonucleotides
used for competition were as follows: VP9,
5`-CCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCG-3` (a gift from Dr. J. M. Redondo,
Hospital de la Princesa, Madrid), containing the two NF-
B-binding
sites from the vascular cell adhesion molecule-1 promoter; KBF,
5`-AGCTTGGGGATTCCCCAT-3`, containing the NF-
B motif of the
promoter of the H-2K
gene (Yano et al., 1987);
AP-1, 5`-CGCTTGATGAGTCAGCCGGAA-3`; and OCT-1,
5`-TGTCGAATGCAAATCACTAGA-3`.
Figure 1: Structure of the 5`-region of the CD69 gene. A, restriction map of the cloned fragment encompassing the 5`-flanking region of the CD69 gene. The restriction sites are BglII (B), EcoRI (E), HindIII (H), PvuII (P), SacI (S), and XbaI (X). The position of the first exon is indicated by a filledbox. B, nucleotide sequence of the 5`-regulatory region of the CD69 gene. First exon nucleotides are underlined, and the major transcription initiation site is denoted by +1. The first intron sequence appears in lower-caseletters. Shadedareas correspond to consensus binding sequences for transcription factors. The TATA box is indicated by doubleunderlining. Prediction of putative transcription factor-binding sites was carried out by the C-coded program SITIOS (Dr. M. A. Vega, Instituto López Neyra, Consejo Superior de Investigaciones Científicas), which includes Release 5.0 of D. Ghosh's transcription factor data base (Ghosh, 1991).
The major transcription initiation site of the CD69 gene has been
located 81 nucleotides upstream from the translation start codon
(Santís et al., 1994). Nucleotide
sequence analysis 1050 bp upstream from the CAP site revealed the
presence of a canonical TATA box at position -30 and a GC-rich
sequence at position -52. In addition, three potential
NF-B/Rel-binding sequences were identified at positions
-160, -223, and -373 (Fig. 1B). The
two proximal NF-
B-binding sites (kB-1 and kB-2 in Fig. 1B) were identical to those found in the
gene promoters of c-myc and interleukin-6, respectively
(Baeuerle, 1991), whereas the most upstream NF-
B motif (kB-3 in Fig. 1B) was similar to that found in the major
histocompatibility complex class I (H-2K
) gene promoter
(Yano et al., 1987).
Figure 2: Functional analysis of the CD69 promoter. The schematic representation of the CD69 promoter-based reporter gene constructs is shown on the left, and the upstream region of the CD69 gene is represented at the top. The positions of the first exon and the first intron are indicated by filled and hatchedboxes, respectively. The nomenclature of the deletion plasmids is based on the most 5`-nucleotide of the CD69 gene sequence present, and its position is denoted relative to the transcription initiation site (position +1). The basal and PMA-induced promoter activity of the 5`-region of the CD69 gene was determined by transient expression of luciferase gene-based constructs in K562 cells. Each transfection was carried out at least four times, and the data from a representative experiment are shown on the right. INDUCT., induction.
Figure 3:
TNF--induced expression of the CD69
antigen is mediated by an increase in the CD69 promoter activity. A, surface expression of the CD69 antigen was analyzed by flow
cytometry on unstimulated K562 cells and on K562 cells treated for 16 h
with PMA (20 µg/ml) or TNF-
(50 ng/ml). Staining of cells with
myeloma P3X63 antibody was included as a negative control. B,
K562 cells were stably transfected with plasmid pAIM1.4-Luc, and the
TNF-
-induced luciferase activity was analyzed in three independent
clones. The luciferase activities are represented in light
units/10
cells. C, activation of the CD69 gene
promoter by the NF-
B/Rel family members. Five µg of construct
pAIM1.4-Luc were cotransfected into K562 cells with 5 or 10 µg of
expression vector pRc/CMV-p50, pRc/CMV-RelA, or pRc/CMV-c-Rel. The
luciferase activities are represented as -fold activation over the
value obtained with construct pAIM 1.4-Luc cotransfected with 10 µg
of empty vector pRc/CMV. Results shown are representative of four
experiments.
It is known that the NF-B/Rel family of transcription factors
plays an important role in the cytokine induction of many cellular and
viral genes (Baeuerle, 1991). To characterize the functional role of
NF-
B-related proteins in the activation of the CD69 gene promoter,
K562 cells were cotransfected with the construct pAIM1.4-Luc and
expression vectors encoding the p50, RelA, and c-Rel members of the
NF-
B/Rel family. As shown in Fig. 3C, p50 was
unable to transactivate the promoter, whereas c-Rel and RelA
efficiently induced the promoter activity (4-5- and
12-18-fold, respectively). Interestingly, cotransfection with a
combination of p50- and RelA-encoding plasmids did not result in
activation of the CD69 promoter (data not shown).
Figure 4:
Activation of the CD69 promoter by
TNF- is achieved through the sequence spanning positions
-255 to -170, which contains the NF-
B-2 motif. A, CD69 promoter-based luciferase plasmids were transfected
into K562 cells, and half of the transfected cells were treated with
TNF-
for 16 h. Each transfection was carried out five times, and a
representative experiment is shown. B, 5 µg of each of the
CD69 promoter-derived constructs were cotransfected into K562 cells
with 10 µg of expression vector pRc/CMV-RelA. Numbers above the bars indicate -fold transactivation over the
values obtained by cotransfection with the empty vector
pRc/CMV.
To confirm this point, K562 cells were cotransfected
with the CD69 promoter constructs and the expression vector
pRc/CMV-RelA. As shown in Fig. 4B, the pattern of
transactivation of the different CD69 promoter fragments by RelA
correlated with the response of these constructs to TNF- (Fig. 4A). Therefore, deletion of the sequences
containing the
B-2 site greatly diminishes the transactivation of
the CD69 promoter by RelA.
Figure 5:
The
B-2 and
B-1 motifs of the proximal CD69 promoter region are
recognized by NF-
B/Rel family members. A, mobility band
shift assays were performed with oligonucleotide probes CD69-
B-1 (lanes 1-5) and CD69-
B-2 (lanes
6-9), which contained the putative NF-
B motifs located
at positions -160 and -223, respectively. Nuclear extracts
from COS-7 cells (lane1) and from COS-7 cells
transfected with pRc/CMV-p50 and pRc/CMV-RelA (lanes
2-9) were incubated with 0.5 ng of each double-stranded
oligonucleotide probe. Competitor oligonucleotides were added at
50-fold molar excess and included oligonucleotide KBF, which contains
the NF-
B motif of the promoter of the H-2K
gene (lanes3 and 7) and the unrelated
oligonucleotides OCT-1 (lanes4 and 8) and
AP-1 (lanes5 and 9). B,
oligonucleotide probes CD69-
B-2 (lanes 1-6) and
CD69-
B-1 (lanes 7-12) were incubated with nuclear
extracts from uninduced K562 cells (lanes1 and 7) and from K562 cells stimulated with PMA (P; lanes2 and 8) or TNF-
(lanes
3-6 and 9-12). Competitor oligonucleotides
were added at 50-fold molar excess and included the specific
competitors (lanes5 and 11),
oligonucleotide VP9 (which contains two NF-
B-binding sites from
the vascular cell adhesion molecule-1 gene promoter) (lanes6 and 12), and the unrelated oligonucleotide
OCT-1 (lanes4 and 10). Specific DNA-protein
complexes (a-d) are indicated. C, nuclear
extracts from uninduced K562 cells (lanes1 and 7) or from K562 cells stimulated with TNF-
(lanes
2-6 and 8-12) were preincubated either with
preimmune serum (PRE; lanes1, 2, 7, and 8) or with antiserum specific to p50 (lanes3 and 9), RelA (lanes4 and 10), c-Rel (lanes5 and 11), or p52 (lanes6 and 12) prior
to the addition of probe CD69-
B-2 (lanes 1-6) or
CD69-
B-1 (lanes 7-12). The DNA-protein complexes
obtained with probe CD69-
B-1 were exposed three times longer than
those obtained with CD69-
B-2 to get a similar intensity of the
bands.
To characterize the NF-B/Rel-related
proteins that bind to the NF-
B sites of the CD69 promoter in
CD69-expressing cells, the two oligonucleotide probes were incubated
with nuclear extracts prepared from untreated K562 cells and from K562
cells treated with either PMA or TNF-
. Four inducible DNA-protein
complexes (a-d) were observed with both oligonucleotide probes when
they were incubated with extracts from PMA- and TNF-
-treated K562
cells (Fig. 5B). The specific DNA-protein complexes
observed with both probes displayed identical electrophoretic mobility,
suggesting that they bind the same nuclear factors. It is interesting
to note that oligonucleotide CD69-
B-1, which bound a lesser amount
of nuclear proteins, contained one mismatch with respect to the
consensus NF-
B-binding site (5`-GGGRNTYYC-3`), whereas
oligonucleotide CD69-
B-2 perfectly matched the consensus sequence
(Baeuerle, 1991). In both cases, the addition of an excess of unlabeled
specific oligonucleotide to the binding reaction completely abolished
the formation of the inducible DNA-protein complexes (Fig. 5B). Similarly, an equal amount of the
heterologous oligonucleotide VP9, which contained two NF-
B-binding
sites from the vascular cell adhesion molecule-1 gene promoter,
efficiently competed the specific complexes (Fig. 5B).
In contrast, the formation of these DNA-protein complexes was not
blocked by the addition of a heterologous competitor that lacked
NF-
B binding sequences (Fig. 5B).
To identify
the nature of the NF-B/Rel family members that bind to the
NF-
B motifs of the CD69 gene promoter, the binding reactions were
preincubated with antiserum specific to p50, RelA, c-Rel, or p52 (Fig. 5C). The anti-p50 antiserum induced the
disappearance or reduced the intensity of complexes b-d. The
anti-RelA antiserum blocked the formation of complexes a and b, whereas
the anti-c-Rel antiserum inhibited only complex c. In contrast, the
anti-p52 antiserum did not interfere with the formation of any of these
complexes. These results indicate that the slower migrating complex
(complex a) corresponds to a homodimer of RelA, which is more easily
detected when using oligonucleotide CD69-
B-2. Then, from top to
bottom, the complexes correspond to p50/RelA (complex b) and p50/c-Rel
(complex c) heterodimers and to a p50 homodimer (complex d). Taken
together, these results confirm that the NF-
B motifs of the
proximal CD69 promoter are capable of binding to inducible
NF-
B/Rel factors, and therefore, they may be involved in the
TNF-
-mediated induction of CD69 gene expression.
The transcription of the CD69 gene appears to be tightly regulated in vivo, as it is almost exclusively expressed at sites were inflammation takes place, suggesting that inflammatory cytokines may participate in the control of the expression of this gene. In this report, we describe the isolation and functional characterization of the human CD69 gene promoter region. We have focused mainly on the identification of the cis-acting sequences and the nuclear factors involved in the inducible expression of the CD69-encoding gene.
Deletion analysis of the 5`-flanking
region of the CD69 gene has allowed the identification of a proximal
fragment of 94 bp (nucleotides -78 to +16) that is
sufficient to govern basal and PMA-induced promoter activity. This
proximal promoter domain contains a canonical TATA box and a GC-rich
sequence that could be a target for Sp1, a ubiquitous and constitutive
transcription factor that binds to the promoter of many genes, and
Egr-1/Krox-24, a zinc-finger transcription factor whose expression can
be induced by various agents, including phorbol esters, and that has
been implicated in the activation of T- and B-lymphocytes (McMahon and
Monroe, 1995; Krämer et al., 1994;
Pérez-Castillo et al., 1993). Since the
synthesis of CD69-encoding mRNA is rapidly induced upon PMA stimulation
of the cells (López-Cabrera et al.,
1993), additional pre-existing transcription factors should mediate the
PMA-induced expression of the CD69 gene to ensure a rapid
transcriptional response. In this context, computer-aided analysis of
the 5`-flanking region of the CD69 gene revealed the presence of three
NF-B motifs (positions -160, -223, and -374) and
at least one putative AP-1-binding site (position -956), which
may cooperate with the proximal cis-acting elements in the
response to phorbol esters.
We demonstrate herewith that TNF-
is capable of inducing the expression of the CD69 antigen and that this
induction is mediated by an increase in the CD69 promoter activity.
Cotransfection experiments with CD69 promoter-derived reporter
constructs and NF-
B-encoding vectors show that this promoter is
transactivated by members of the NF-
B/Rel family, specially by
RelA (formerly p65). Thus, the TNF-
-induced expression of the CD69
gene may be mediated by the binding of NF-
B/Rel proteins to one or
more NF-
B motifs of the CD69 promoter. This point is further
supported by the fact that deletion of the sequence that contains the
B-2 motif (nucleotides -255 to -170) completely
abolishes the response to TNF-
and markedly reduces RelA-mediated
transactivation. Since no additional TNF-
-responsive elements are
present in this sequence, our results strongly indicate that the
B-2 motif plays a key role in the TNF-
-induced promoter
activity. However, to define unambiguously the contribution of
B-2
and the other NF-
B motifs to the response to TNF-
, we are
currently performing site-directed mutagenesis of these motifs in the
context of the intact promoter.
Our results also demonstrate that
the two most proximal NF-B motifs of the CD69 promoter (
B-2
and
B-1) bind four PMA- and TNF-
-inducible
NF-
B/Rel-related complexes. The higher affinity of these proteins
for the
B-2 sequence further emphasizes the functional role of
this motif and may explain why constructs containing only the
B-1
site are unresponsive to TNF-
. Antisera directed against the
different NF-
B/Rel proteins were used to identify the family
members present in the DNA-protein complexes detected with the
NF-
B motif-derived probes. These experiments revealed that the
DNA-binding activities consisted of a RelA homodimer, p50/RelA and
p50/c-Rel heterodimers, and a p50 homodimer. The most prominent
complexes were composed of p50/RelA and p50/c-Rel heterodimers. The
detection of RelA homodimer binding to the
B-2 motif, a complex
that is not easily detected with the NF-
B sites of other promoters
(Ganchi et al., 1993), is consistent with the observation that
the sequence of this motif matches perfectly with the consensus binding
sequence for RelA, 5`-GGGRNTTTCC-3` (Kunsch et al., 1992).
We have recently demonstrated that the expression of the CD69 gene
is regulated at the post-transcriptional level by a rapid degradation
pathway associated with AU-rich sequence motifs
(Santís et al., 1995). This
mechanism of regulation has been found in many genes involved in
inflammatory and activation responses. Most of these genes code for
cytokines and oncoproteins, which are implicated in the initial events
leading to activation and proliferation of the cells. In addition, the
transcription of these genes appears to be highly regulated by the
NF-B motifs of their promoters (Baeuerle, 1991; Baeuerle and
Henkel, 1994). Therefore, our findings, which highlight the functional
relevance of the NF-
B motifs of the CD69 gene, further support the
idea regarding a general mechanism involved in the control of
expression of activation-associated genes during the early phase of the
immune response.
The expression of CD69-encoding mRNA can be easily
induced in most leukocytes by treatment ``in vitro''
with a wide range of stimuli such as PMA or anti-CD3/T cell receptor
mAb (López-Cabrera et al., 1993; Ziegler et al., 1994). In addition, the expression of CD69 on
activated T-lymphocytes from cell infiltrates of various chronic
inflammatory diseases such as rheumatoid arthritis and chronic viral
hepatitis has been documented
(García-Monzón et al., 1990; Laffón et al.,
1991). It is also known that TNF- is an important inflammatory
mediator that is actively secreted by several cell types at
inflammatory sites (González-Amaro et
al., 1994; Vassalli, 1992). Our findings strongly suggest that the
pro-inflammatory cytokine TNF-
may play a key role ``in
vivo'' in the expression of CD69 by inflammatory cells.
Interestingly enough, we have previously described that CD69 antigen is
in turn capable of generating signals that induce the synthesis of
TNF-
by lymphoid cells (Santís et
al., 1992). Thus, it is feasible that, at sites of inflammation, a
positive feed-back loop is established between the expression of CD69
and the production of TNF-
. The putative ligand of CD69 should be
clearly involved in this condition. The above phenomenon could have an
important role in the perpetuation of several inflammatory diseases and
could be related to the resistance of some patients with these
conditions toward the current anti-inflammatory therapy.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z38109[GenBank].