TCA3-Mouse CCL1

8 Pages·2001·0.121 MB·English

by  Dorf M.A.

TCA3/Mouse CCL1
Martin E. Dorf
Department of Pathology, Harvard Medical School, 200 Longwood Avenue,
Boston, MA 02115, USA
corresponding author tel: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2001.1104.
Chapter posted 5 November 2001
SUMMARY Alternative names
TCA3 is a mouse CC chemokine that mediates TCA3isanacronymforTcellactivationgene3.The
chemotaxis and activation of neutrophils and macro- proposed human homolog of TCA3 is termed I-309
phages. TCA3 also affects the growth and survival of (Milleretal.,1989).Inthelatestclassificationsystem,
some T lymphocytes, mesangial and vascular smooth TCA3 and I-309 were renamed CCL1 (Zlotnik and
musclecells.TCA3expressionisrestrictedtoantigen- Yoshie, 2000).
activated T cells and mast cells. It is a high-affinity
ligandforthechemokinereceptor CCR8plusatleast
one additional receptor yet to be identified.
Structure
The homologies between TCA3 and mouse MCP-1/
CCL2 were initially used to propose the CC(X) –
BACKGROUND 22-24
C(X) C motif now characteristic of most CC
13-15
chemokines (Burd et al., 1988). The initiation codon
Discovery
(ATG) in TCA3 is followed by a region encoding
hydrophobic aminoacids, typical ofthesignal peptide
Subtractive hybridization was used to identify mouse required for protein secretion (Burd et al., 1988). A
T cell-specific genes transcribed during the activation signal peptide cleavage site is located between amino
process. One previously undefined T cell activation- acids S23 and K24 of the unprocessed protein.
specificgenewastermedTCA3.Thisgeneencodesan One site located within a predicted turn region is
mRNA that is expressed within one hour following present for potential N-linked glycosyation (Figure 1).
Concanavalin A activation of T cell clones reaching RecombinantTCA3canbecomeunstableafterstorage
levels of approximately 1% of the total poly(A)- at (cid:255)80(cid:14)C for over 6 months. Although TCA3 and I-
containingmRNA(Burdetal.,1987).TCA3message 309showonly42%homologyinaminoacidsequence,
wasnotdetectedinmRNAfromunstimulatedTcells, both proteins share several biochemical similarities.
resting or LPS-activated B cells, or RNA from Most interestingly, TCA3 and I-309 possess an extra
thymus, brain, heart, liver, lung, spleen, and kidney. pairofcysteines,whichputativelystabilizetheposition
Induction of TCA3 transcripts in T lymphocytes oftheC-terminalportionofthemolecule(Keizeretal.,
required either antigen or mitogen activation. 2000). The native TCA3 and I-309 gene products are
Stimulation of T cells with the T cell growth factor, secretedglycoproteinswithapparentmolecularweights
IL-2, failed to induce TCA3 transcripts (Burd et al., of 15–17kDa, twice that predicted for the polypeptide
1987). backboneoftheTCA3protein(Milleretal.,1989;Luo
CytokineReference Copyright#2001AcademicPress
2 Martin E. Dorf
Figure 1 Predicted sequence of mature secreted TCA3 and P500 proteins
after removal of signal peptide sequence (MKPTAMALMCLLLAAV-
WIQDVDS). The four cysteine (C) residues comprising the classical
chemokine motif are indicated in underlined, the N-linked glycosylation
motif is shown in bold.

TCA3
KSMLTVSNSCCLNTLKKELPLKFIQCYRKMGSSCPDPPAVVFRLNKGRESCASTNKTWVQNHLKKVNPC
– – –
KSMLTVSNSCCLNTLKKELPLKFIQCYRKMGSSCPDPPAVVFRSSGVPGLTEAEKTVHRFQ
– –
P500
et al., 1993). Subsequently, Brown et al. (1989) Human gene: M57506
described a murine cDNA clone designated P500 Chicken cDNA: L34552
whose nucleotide sequence was identical to TCA3 Inaddition,cDNAforratTCA3hasbeenpartially
through 260 nucleotides, but diverged thereafter. As sequenced. It displayed 81% homology with mouse
detailed subsequently, P500 represents an alternative TCA3 (Natori et al., 1997). Like the alternate splice
splice variant of TCA3. product P500, the putative chicken TCA3 sequence
lacks one of the highly conserved cysteine residues
used to form the 2–4 disulfide bridge implicated in
Main activities and
chemokine folding.
pathophysiological roles
Sequence
Subcutaneousinjectionof3nMLPS-freerecombinant
TCA3 into normal mice caused a rapid swelling
response characterized histologically by massive local The TCA3 cDNA and genomic sequences are
accumulation of neutrophils (Wilson et al., 1990b). available at GenBank (http://www.ncbi.nlm.nih.gov).
Intraperitoneal injection of 10–500ng HPLC purified
rTCA3 resulted in an influx of neutrophils into the
peritoneum within 2 hours. In addition, i.p. injection Chromosomal location
of TCA3 induced a smaller but statistically significant
increase of macrophages (Luo et al., 1994). There was
The locus encoding TCA3 is termed Scya1 and
littleeffectwithlowerdosesofrTCA3.Theneutrophils
was mapped using interspecies somatic cell hybrids
and monocytes were recruited from the blood where
and recombinant inbred mouse strains to the distal
similar changes in cellular composition could be
portion of mouse chromosome 11 near the Hox-2
detected within 15–45 minutes (Luo et al., 1994). The
gene complex (Wilson et al., 1990a). Like most other
kinetics of the neutrophil and monocyte changes are
CC chemokines TCA3 consists of three exons and
identical, suggesting that TCA3 acts simultaneously
two introns, but has two splice acceptor sites in the
on both populations rather than acting sequentially.
second intron. The alternative splicing generates
The specificity of this in vivo reaction was demon-
a second transcript, termed P500, with a distinct
strated by complete inhibition with neutralizing anti-
50 boundaryforexon3resultingin99additionalbase
TCA3 antibodies (Luo et al., 1993, 1994). TCA3
pairsinthemiddleoftheRNAcomparedwithTCA3
overexpression in mice resulted in enhanced tumor
(Wilson et al., 1990a). This results in dramatically
immunity (Laning et al., 1994) and TCA3 also shows
different amino acids from position 64 to the end
immuno-adjuvant activity (Tsuji et al., 1997).
of the proteins. Most notably, P500 lacks a critical
highly conserved cysteine corresponding to cysteine
position 4 that is present in all other chemokines.
GENE AND GENE REGULATION
Additional TCA3 splice variants involving deletion
ormodificationofthelast threeaminoacidsencoded
Accession numbers
by exon 2 (residues 41–43) have also been reported
(Kennedy et al., 2000). TCA3 and I-309 have
Mouse cDNA: M17957 extensive homology around the alternative intron
Mouse gene: X52401 splice acceptor site, suggesting that humans may also
Human cDNA: P22362 utilize both splice sites and may have a product
TCA3/Mouse CCL1 3
homologoustoP500(Milleretal.,1990;Wilsonetal., Cells and tissues that express
1990a).
the gene
Relevant linkages Most activated T lymphocytes or mast cells are
known to synthesize mouse TCA3 transcripts
(Kuchroo et al., 1993). TCA3 transcription is rapidly
The gene encoding TCA3 mapped in a cluster with
inducedafteractivationofmurineTH1,TH2,orCTL
other chemokine genes, including those that encode
clones and NK T cells (Kennedy et al., 2000; Wilson
MCP-1 (Scya2), MIP-1(cid:11) (Scya3), and MIP-1(cid:12)
et al., 1988). Mitogen-activated naı¨ve splenocytes
(Scya4) (Wilson et al., 1990a).
also produce TCA3 transcripts (Wilson et al., 1988).
Polymorphisms for TCA3 exist within the coding
When the northern blots initially used to identify
sequence. Divergent sequences between the Balb or
TCA3 mRNA were lightly exposed, a second distinct
C57BL and SJL inbred mouse strains result in
band was often revealed. The two bands were of
alterations of four amino acids in the C-terminal
similar molecular size but the signal from one band
region of TCA3 (Teuscher et al., 1999). These
was so intense it obscured identification of the minor
polymorphisms are linked to the disease-modifying
band. Brown et al. (1989) first identified a transcript
loci that control development of monophasic remit-
containing a possible alternate splice of TCA3,
ting/nonrelapsing experimental allergic encephalo-
termed P500. Subsequent analysis revealed the ratio
myelitis (Teuscher et al., 1999).
of TCA3:P500 transcripts ranged between 5:1 and
10:1 in T cells while the ratio ranged between 0.1:1
and 0.5:1 in mast cells (Laning, 1995). TCA3 is the
Regulatory sites and corresponding
most abundant activation-specific gene detected in
transcription factors NKT cells. However, P500 transcripts were not
reported among 100 randomly selected clones from
Comparisons of the 50 untranslated sequences for an activation-specific NKT subtraction library
TCA3 ((cid:255)320 to (cid:255)76) and its human homolog I-309 (Kennedy et al., 2000).
((cid:255)314 to (cid:255)91) demonstrated a 69% nucleotide
sequence identity. This region includes several highly
conserved motifs that appear to be important for
PROTEIN
regulation of transcription. These include a palin-
dromic NF(cid:20)B-related sequence, the PU.1 myeloid-
Accession numbers
lymphoid specific transcriptional activator, and Py
the polyoma early enhancer core sequence (Miller
et al., 1990). SwissProt:
Regulation of the TCA3 gene was examined in Mouse: P10146
mast cells. Mast cells produce TCA3 by de novo Human: P22362
transcription following crosslinking of IgE receptors
or after pharmacological treatment with PMA plus
Sequence
a calcium ionophore (Burd et al., 1989). Using mast
cells transfected with TCA3 promoter CAT con-
structs, it was shown that inducible expression is The TCA3 amino acid sequence is available
directed by a region extending 82bp upstream from at GenBank and Protein Data Bank (http://
the transcription start site. Transcription was www.ncbi.nlm.nih.gov).
enhanced by a region extending further to 1324kb Refer to Figure 1 for sequence comparisons
upstream (Oh and Metcalfe, 1994). The TCA3 between the alternate splice products of TCA3.
gene has a functional NF(cid:20)B element at position
(cid:255)194 to (cid:255)185 (Oh et al., 1997). In addition, two
Description of protein
negative regulatory regions were identified, NRE-1
andNRE-2.BothNRE-1andNRE-2independently
inhibit CAT synthesis. Electrophoretic mobility Native TCA3 secreted from activated T cell clones
shift assays were used to identify the putative appears as a single band at 14–15kDa by western
silencer regions. Two sequences within each NRE blot. Although rTCA3 resolved as a single peak
were identified, including one novel silencer motif by HPLC it ran as 2–4 bands by SDS-PAGE. Five
(Oh et al., 1997). individual peaks were detected when rTCA3 was
4 Martin E. Dorf
analyzed by mass spectrometry with calculated mole- paralleled the IFN(cid:13) transcription pattern. In addi-
cular weights of 9.2–10.2kDa with a predominant tion,anti-CD3-activatedTCR(cid:11)(cid:12)(cid:135),NK1.1(cid:135),CD4(cid:255),
peak at 9827 (Luo et al., 1994). Thus, the 14–17kDa CD8(cid:255), NK-T cells make high levels of TCA3
apparent molecular weight noted by SDS-PAGE is mRNA (Kennedy et al., 2000). The signals that
an overestimate. HPLC-purified rTCA3 contains a induce TCA3 and other cytokines can be dissociated
homogeneous peptide as verified by sequencing the among individual T cell clones (Kuchroo et al., 1993;
N-terminal 30 amino acids which contained the Wilson et al., 1988). Furthermore, TCA3 transcrip-
predicted TCA3 sequence (Luo et al., 1994). Thus, tion marks T cell activation even in the absence of
the heterogeneity noted in rTCA3 glycoproteins a proliferative response (Wilson et al., 1988). TCA3
probably represents glycosylation differences of the was not induced following IL-2 stimulation (Burd
TCA3 monomer. The crystal structure of TCA3 et al., 1987). Stimulation through the T cell receptor
has not been resolved but the three-dimensional can be bypassed by simultaneous pharmacologic
solution structure of its human homolog, I-309, was activation of protein kinase C and increase of
determined by nuclear magnetic resonance spectros- intracellular calcium (Wilson et al., 1988). The data
copy and dynamic simulated annealing (Keizer suggest that TCA3 is induced by activation through
et al., 2000). While the general fold of nonglyco- the TCR, but not by activation via the IL-2 receptor.
sylated synthetic I-309 conformed to that of other Like many other inducible lymphokines, expression
CC chemokines, significant differences were intro- of TCA3 mRNA was completely blocked by
duced to the C-terminal (cid:11) helix by the third disulfide cyclosporin A treatment (Burd et al., 1987).
bond common to TCA3 and I-309. Mast cells or mast cell lines are induced to
transcribe TCA3 in response to crosslinking of IgE
receptors by IgE and specific multivalent antigens.
Important homologies
In selected instances TCA3 expression can also be
induced by activation of mast cells with Con A or
Based on amino acid sequence homology, the most phorbol ester plus ionophore (Burd et al., 1989). An
significant homology of TCA3 is with human I-309 alternative mast cell activation pathway capable of
(42% overall protein identity). This is considerably triggering TCA3 mRNA expression may also exist
less homology than has been found among other (Talkington and Nickell, 2001). Stimulation of mast
chemokine pairs. cells with the growth factor IL-3, failed to induce
TCA3 transcripts (Burd et al., 1989).
CELLULAR SOURCES AND
RECEPTOR UTILIZATION
TISSUE EXPRESSION
TCA3 binds with high (2nM) affinity to the mouse
Cellular sources that produce
chemokine receptor CCR8; I-309 partially inhibits
thisinteraction(Goyaetal.,1998).CCR8isprimarily
Mouse TCA3 has a highly restricted cell distribution.
expressedonasubsetofthymocytesandonactivated
Most antigen- or mitogen-activated T cell clones
polarized TH2 cells (Kremer et al., 2001; Zingoni
synthesize mouse TCA3 proteins. Mitogen-activated
et al., 1998). Mouse macrophages, mouse mesangial
naı¨ve splenocytes also produce TCA3 proteins but at
cells, and rat vascular smooth muscle cells that do
lower levels than T cell clones (Luo et al., 1993). notexpressCCR8alsobind125I-TCA3withasimilar
K value (3–4nM) (Luo et al., 1996; Luo and Dorf,
d
1996; Goya et al., 1998). Thus, one or more addi-
Eliciting and inhibitory stimuli,
tional chemokine receptors can bind TCA3.
including exogenous and
endogenous modulators
IN VITRO ACTIVITIES
TCA3 is expressed as an early cell activation gene.
In vitro findings
TCA3 or P500 mRNA can be detected as early as
1hourafterTcellactivationwithantigenormitogen.
mRNA accumulates to an apparent peak level by TCA3 displays chemoattractant and activating acti-
4 hours in antigen-activated T cells then decreases vities on a variety of hematopoietic and nonhema-
in abundance (Wilson et al., 1988). These profiles topoietic target cells (Table 1). The chemoattractant
TCA3/Mouse CCL1 5
Table 1 In vitro activities of TCA3
Target cells Biological activity Concentration References
Neutrophils Cell adhesion 10(cid:255)11–10(cid:255)9M Devi et al., 1995
Chemoattractant 10(cid:255)9–10(cid:255)7M Luo et al., 1994
Nitrite production 10(cid:255)8–10(cid:255)7M Devi et al., 1995
O (cid:255) production 10(cid:255)8–10(cid:255)7M Devi et al., 1995
2
H O production 10(cid:255)8–10(cid:255)7M Devi et al., 1995
2 2
Granule exocytosis 10(cid:255)7M Devi et al., 1995
Macrophage/monocyte Cell adhesion 10(cid:255)11–10(cid:255)9M Devi et al., 1995
Chemoattractant 10(cid:255)8–10(cid:255)7M Luo et al., 1994
Nitrite production 10(cid:255)8–10(cid:255)7M Devi et al., 1995
Calcium mobilization 10(cid:255)8M Luo et al., 1994
Granule exocytosis 10(cid:255)11–10(cid:255)8M Devi et al., 1995
T lymphocytes Chemoattractant 10(cid:255)12–10(cid:255)7M Zingoni et al., 1998
Anti-apoptotic 10(cid:255)10M Van Snick et al., 1996
Mesangial cells Cell adhesion 10(cid:255)11–10(cid:255)8M Luo et al., 1994
Chemoattractant 10(cid:255)9–10(cid:255)7M Luo et al., 1994
Growth/survival 10(cid:255)8M Luo et al., 1994
Vascular smooth muscle Cell adhesion 10(cid:255)11–10(cid:255)8M Luo et al., 1996
Chemotaxis 10(cid:255)10–10(cid:255)7M Luo et al., 1996
Growth/survival 10(cid:255)8M Luo et al., 1996
Microglia Chemoattractant 10(cid:255)10–10(cid:255)8M Hayashi et al., 1995
Astrocytes Chemoattractant 10(cid:255)8M Heesen et al., 1996
and activating capacities are coordinated. At the no lymphocyte or neutrophil involvement (Laning,
lowest concentrations of TCA3, when very few 1995).
receptors are engaged, cell adhesion to matrix pro-
teins increases and cells begin to migrate. As the
Bioassays used
target cells reach the focus of TCA3 production,
where the TCA3 concentration should be the
highest, phagocytic target cells become activated to The most common and one of the most sensitive
degranulate and release proteolytic enzymes and assaysusedforTCA3ischemotaxis.Theglycosylated
otherpotentiallytissue-destructiveagents(Devietal., and nonglycosylated forms of rTCA3 have similar
1995). In all cases tested TCA3-induced responses chemotactic properties (Laning, 1995).
are sensitive to pertussis toxin treatment, indicating
a critical role for G protein-coupled receptors
(cid:11)i
in TCA3-mediated signal transduction. TCA3 also IN VIVO BIOLOGICAL
induced anti-apoptotic activity affecting the survival
ACTIVITIES OF LIGANDS IN
and growth of some T lymphocytes and possibly
ANIMAL MODELS
non-hematopoietic cells (Table 1).
In vitro P500 is a chemoattractant for macro-
phages, monocytic cell lines, and microglia (Laning, J558 myeloma cells were transfected with an
1995). Intraperitoneal injection of P500 also induces expression vector for TCA3 to study the pathophy-
a rapid inflammatory response in vivo characterized siological effects of TCA3 overproduction (Laning
by a predominant monocytic infiltrate with little or et al., 1994). For tumor transplantation, transfected
6 Martin E. Dorf
or control cells were injected subcutaneously into the results imply that TCA3 can act as an adjuvant
flank of syngeneic Balb/c mice. The control and by recruiting antigen-presenting cells and enhancing
vector transfected tumors grew progressively at the cell-mediated immunity.
same rate, but only 11% of tumors in the TCA3-
transfected group continued to progress (Laning
et al., 1994). Some of the surviving recipients of the
PATHOPHYSIOLOGICAL ROLES
TCA3-transfected cells were challenged with non-
IN NORMAL HUMANS AND
transfected control J558 cells on the opposite flank;
none of these mice developed a detectable tumor DISEASE STATES AND
mass during the 23-day observation period, while
DIAGNOSTIC UTILITY
all naı¨ve recipients developed progressively growing
tumor masses. Reciprocal experiments with a second
Role in experiments of nature and
TCA3-transfected Balb/c myeloma, P3X, were per-
formed to demonstrate tumor specificity. TCA3- disease states
transfected P3X cells provided protection to chal-
lenge with normal P3X cells, but not against J558.
Chemokines have been associated with the develop-
Similarly mice that recovered from the TCA3-
ment of several cell-mediated autoimmune diseases.
transfected J558 tumors were completely susceptible
Expression of TCA3 transcripts were first correlated
to P3X (Laning et al., 1994). The immunity was
with the encephalitogenic potency of T cell clones
TCA3-dependent, as priming with irradiated tumor
(Kuchroo et al., 1993). These findings were sup-
cells provided little protection against challenge
ported by the observation that TCA3 mRNA was
with nontransfected tumor cells. Mixing TCA3-
specifically induced in spinal cord 1–2 days before
transfected cells with normal tumor cells caused
clinical signs of experimental allergic encephalo-
retarded growth of the normal tumor cells, provided
myelitis (EAE) were apparent (Godiska et al.,
the latter were injected into the same site. Further-
1995). Another study demonstrated the correlation
more, direct in situ injection of TCA3 early during
of TCA3 upregulation with enhanced neutrophilia
the course of tumor implantation also inhibited
inEAE(Tranetal.,2000).TheexpressionofCCR8,
tumor growth (Laning et al., 1994). Histologic ex-
a TCA3 receptor, also correlates with the develop-
amination of the TCA3-transfected tumor
ment of inflammatory lesions in EAE (Fischer et al.,
injection site identified infiltrating neutrophil and
2000).
macrophage cell populations without noticeable
Administration of anti-TCA3 monoclonal anti-
accumulation of lymphocytes. The combined data
body to cryptococcal immune mice directly demon-
suggest that TCA3 stimulates or augments tumor
strated the role of TCA3 in protection against this
immunogenicity perhaps by attracting phagocytic
fungal pathogen. Anti-TCA3 treatment also reduced
cells that facilitate tumor destruction and presenta-
TH1-mediated DTH responses to cryptococcal anti-
tion of tumor antigens to the immune system. Thus,
gens and influenced the migration of leukocytes into
TCA3 was viewed as a biological adjuvant (Laning
the DTH reaction site (Doyle and Murphy, 1999).
et al., 1994).
Anti-TCA3 treatments reduced both the number of
To test the concept that TCA3 could serve as an
neutrophils entering the DTH site and the amount
adjuvant for cell-mediated immunity, a TCA3
of the T cell chemoattractant, MIP-1(cid:11), that the
expression plasmid containing a muscle-specific
neutrophils produced (Doyle and Murphy, 1999).
promotor was co-injected into muscle along with an
Thus, TCA3 was indirectly involved in recruiting
HIV-specificDNAvaccine.Histologicanalysisofthe
lymphocytes into the DTH reaction. As EAE is also
injection site revealed predominance of infiltrating
mediated by TH1 cells and is associated with
mononuclear cells with the TCA3 plasmid. In
expression of TCA3 and MIP-1(cid:11); this chemokine
contrast, no accumulation of inflammatory cells was
networkmodelmayalsoapplytothe developmentof
detected in controls (Tsuji et al., 1997). Addition of
cell-mediated autoimmune diseases.
TCA3 also enhanced delayed-type hypersensitivity
(DTH) and cytolytic T cell (CTL) responses (Tsuji
et al., 1997). The effects were TCA3-specific as
ACKNOWLEDGEMENTS
administration of anti-TCA3 suppressed DTH
responses. Increases in HIV-specific IgG2a titers
suggested that inoculation of the TCA3 plasmid This work was partially supported by NIH grant CA
skewedtheresponsetowardTH1cells.Thecombined 67416.
TCA3/Mouse CCL1 7
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Zingoni,A.,Soto,H.,Hedrick,J.A.,Stoppacciaro,A.,Storlazzi,
C. T., Sinigaglia, F., D’ambrosio, D., O’Garra, A., Robinson,
D., Rocchi, M., Santoni, A., Zlotnik, A., and Napolitano, M. See Table 2.
Table 2 Sources for TCA3 reagents
Product Commercial supplier Catalog number Uses
Recombinant TCA3 BD PharMingen 19351V Chemotaxis, Bioassays
R & D Systems 845-TC-025
Polyclonal anti-TCA3 R & D Systems AF845 Neutralization, Western Blot
Monoclonal anti-TCA3 BD PharMingen 18230D Neutralization, Western Blot
ELISA Set BD PharMingen 2669KI Protein Quantitation
mRNA Template Sets BD PharMingen 45026P RNase Protection Assay