TCA3/Mouse CCL1 Martin E. Dorf Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA corresponding author tel: 617-432-1978, fax: 617-432-2789, 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 chemotaxis and activation of neutrophils and macrophages. TCA3 also affects the growth and survival of some T lymphocytes, mesangial and vascular smooth muscle cells. TCA3 expression is restricted to antigenactivated T cells and mast cells. It is a high-affinity ligand for the chemokine receptor CCR8 plus at least one additional receptor yet to be identified.
TCA3 is an acronym for T cell activation gene 3. The proposed human homolog of TCA3 is termed I-309 (Miller et al., 1989). In the latest classification system, TCA3 and I-309 were renamed CCL1 (Zlotnik and Yoshie, 2000).
BACKGROUND
Discovery Subtractive hybridization was used to identify mouse T cell-specific genes transcribed during the activation process. One previously undefined T cell activationspecific gene was termed TCA3. This gene encodes an mRNA that is expressed within one hour following Concanavalin A activation of T cell clones reaching levels of approximately 1% of the total poly(A)containing mRNA (Burd et al., 1987). TCA3 message was not detected in mRNA from unstimulated T cells, resting or LPS-activated B cells, or RNA from thymus, brain, heart, liver, lung, spleen, and kidney. Induction of TCA3 transcripts in T lymphocytes required either antigen or mitogen activation. Stimulation of T cells with the T cell growth factor, IL-2, failed to induce TCA3 transcripts (Burd et al., 1987).
Cytokine Reference
Structure The homologies between TCA3 and mouse MCP-1/ CCL2 were initially used to propose the CC(X)22-24± C(X)13-15C motif now characteristic of most CC chemokines (Burd et al., 1988). The initiation codon (ATG) in TCA3 is followed by a region encoding hydrophobic amino acids, typical of the signal peptide required for protein secretion (Burd et al., 1988). A signal peptide cleavage site is located between amino acids S23 and K24 of the unprocessed protein. One site located within a predicted turn region is present for potential N-linked glycosyation (Figure 1). Recombinant TCA3 can become unstable after storage at ÿ80 C for over 6 months. Although TCA3 and I309 show only 42% homology in amino acid sequence, both proteins share several biochemical similarities. Most interestingly, TCA3 and I-309 possess an extra pair of cysteines, which putatively stabilize the position of the C-terminal portion of the molecule (Keizer et al., 2000). The native TCA3 and I-309 gene products are secreted glycoproteins with apparent molecular weights of 15±17 kDa, twice that predicted for the polypeptide backbone of the TCA3 protein (Miller et al., 1989; Luo
Copyright # 2001 Academic Press
2
Martin E. Dorf Figure 1 Predicted sequence of mature secreted TCA3 and P500 proteins after removal of signal peptide sequence (MKPTAMALMCLLLAAVWIQDVDS). 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) described a murine cDNA clone designated P500 whose nucleotide sequence was identical to TCA3 through 260 nucleotides, but diverged thereafter. As detailed subsequently, P500 represents an alternative splice variant of TCA3.
Main activities and pathophysiological roles Subcutaneous injection of 3 nM LPS-free recombinant TCA3 into normal mice caused a rapid swelling response characterized histologically by massive local accumulation of neutrophils (Wilson et al., 1990b). Intraperitoneal injection of 10±500 ng HPLC purified rTCA3 resulted in an influx of neutrophils into the peritoneum within 2 hours. In addition, i.p. injection of TCA3 induced a smaller but statistically significant increase of macrophages (Luo et al., 1994). There was little effect with lower doses of rTCA3. The neutrophils and monocytes were recruited from the blood where similar changes in cellular composition could be detected within 15±45 minutes (Luo et al., 1994). The kinetics of the neutrophil and monocyte changes are identical, suggesting that TCA3 acts simultaneously on both populations rather than acting sequentially. The specificity of this in vivo reaction was demonstrated by complete inhibition with neutralizing antiTCA3 antibodies (Luo et al., 1993, 1994). TCA3 overexpression in mice resulted in enhanced tumor immunity (Laning et al., 1994) and TCA3 also shows immuno-adjuvant activity (Tsuji et al., 1997).
GENE AND GENE REGULATION
Accession numbers Mouse cDNA: M17957 Mouse gene: X52401 Human cDNA: P22362
Human gene: M57506 Chicken cDNA: L34552 In addition, cDNA for rat TCA3 has been partially sequenced. It displayed 81% homology with mouse TCA3 (Natori et al., 1997). Like the alternate splice 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 chemokine folding.
Sequence The TCA3 cDNA and genomic sequences are available at GenBank (http://www.ncbi.nlm.nih.gov).
Chromosomal location The locus encoding TCA3 is termed Scya1 and was mapped using interspecies somatic cell hybrids and recombinant inbred mouse strains to the distal portion of mouse chromosome 11 near the Hox-2 gene complex (Wilson et al., 1990a). Like most other CC chemokines TCA3 consists of three exons and two introns, but has two splice acceptor sites in the second intron. The alternative splicing generates a second transcript, termed P500, with a distinct 50 boundary for exon 3 resulting in 99 additional base pairs in the middle of the RNA compared with TCA3 (Wilson et al., 1990a). This results in dramatically different amino acids from position 64 to the end of the proteins. Most notably, P500 lacks a critical highly conserved cysteine corresponding to cysteine position 4 that is present in all other chemokines. Additional TCA3 splice variants involving deletion or modification of the last three amino acids encoded by exon 2 (residues 41±43) have also been reported (Kennedy et al., 2000). TCA3 and I-309 have extensive homology around the alternative intron splice acceptor site, suggesting that humans may also utilize both splice sites and may have a product
TCA3/Mouse CCL1 3 homologous to P500 (Miller et al., 1990; Wilson et al., 1990a).
Cells and tissues that express 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 induced after activation of murine TH1, TH2, or CTL clones and NK T cells (Kennedy et al., 2000; Wilson et al., 1988). Mitogen-activated naõÈ ve splenocytes also produce TCA3 transcripts (Wilson et al., 1988). When the northern blots initially used to identify TCA3 mRNA were lightly exposed, a second distinct band was often revealed. The two bands were of similar molecular size but the signal from one band was so intense it obscured identification of the minor band. Brown et al. (1989) first identified a transcript containing a possible alternate splice of TCA3, termed P500. Subsequent analysis revealed the ratio 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 most abundant activation-specific gene detected in NKT cells. However, P500 transcripts were not reported among 100 randomly selected clones from an activation-specific NKT subtraction library (Kennedy et al., 2000).
The gene encoding TCA3 mapped in a cluster with other chemokine genes, including those that encode MCP-1 (Scya2), MIP-1 (Scya3), and MIP-1 (Scya4) (Wilson et al., 1990a). Polymorphisms for TCA3 exist within the coding sequence. Divergent sequences between the Balb or C57BL and SJL inbred mouse strains result in alterations of four amino acids in the C-terminal region of TCA3 (Teuscher et al., 1999). These polymorphisms are linked to the disease-modifying loci that control development of monophasic remitting/nonrelapsing experimental allergic encephalomyelitis (Teuscher et al., 1999).
Regulatory sites and corresponding transcription factors Comparisons of the 50 untranslated sequences for TCA3 (ÿ320 to ÿ76) and its human homolog I-309 (ÿ314 to ÿ91) demonstrated a 69% nucleotide sequence identity. This region includes several highly conserved motifs that appear to be important for regulation of transcription. These include a palindromic NFB-related sequence, the PU.1 myeloidlymphoid specific transcriptional activator, and Py the polyoma early enhancer core sequence (Miller et al., 1990). Regulation of the TCA3 gene was examined in mast cells. Mast cells produce TCA3 by de novo transcription following crosslinking of IgE receptors or after pharmacological treatment with PMA plus a calcium ionophore (Burd et al., 1989). Using mast cells transfected with TCA3 promoter CAT constructs, it was shown that inducible expression is directed by a region extending 82 bp upstream from the transcription start site. Transcription was enhanced by a region extending further to 1324 kb upstream (Oh and Metcalfe, 1994). The TCA3 gene has a functional NFB element at position ÿ194 to ÿ185 (Oh et al., 1997). In addition, two negative regulatory regions were identified, NRE-1 and NRE-2. Both NRE-1 and NRE-2 independently inhibit CAT synthesis. Electrophoretic mobility shift assays were used to identify the putative silencer regions. Two sequences within each NRE were identified, including one novel silencer motif (Oh et al., 1997).
PROTEIN
Accession numbers SwissProt: Mouse: P10146 Human: P22362
Sequence The TCA3 amino acid sequence is available at GenBank and Protein Data Bank (http:// www.ncbi.nlm.nih.gov). Refer to Figure 1 for sequence comparisons between the alternate splice products of TCA3.
Description of protein Native TCA3 secreted from activated T cell clones appears as a single band at 14±15 kDa by western blot. Although rTCA3 resolved as a single peak by HPLC it ran as 2±4 bands by SDS-PAGE. Five individual peaks were detected when rTCA3 was
4
Martin E. Dorf
analyzed by mass spectrometry with calculated molecular weights of 9.2±10.2 kDa with a predominant peak at 9827 (Luo et al., 1994). Thus, the 14±17 kDa apparent molecular weight noted by SDS-PAGE is an overestimate. HPLC-purified rTCA3 contains a homogeneous peptide as verified by sequencing the N-terminal 30 amino acids which contained the predicted TCA3 sequence (Luo et al., 1994). Thus, the heterogeneity noted in rTCA3 glycoproteins probably represents glycosylation differences of the TCA3 monomer. The crystal structure of TCA3 has not been resolved but the three-dimensional solution structure of its human homolog, I-309, was determined by nuclear magnetic resonance spectroscopy and dynamic simulated annealing (Keizer et al., 2000). While the general fold of nonglycosylated synthetic I-309 conformed to that of other CC chemokines, significant differences were introduced to the C-terminal helix by the third disulfide bond common to TCA3 and I-309.
Important homologies Based on amino acid sequence significant homology of TCA3 (42% overall protein identity). less homology than has been chemokine pairs.
homology, the most is with human I-309 This is considerably found among other
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce Mouse TCA3 has a highly restricted cell distribution. Most antigen- or mitogen-activated T cell clones synthesize mouse TCA3 proteins. Mitogen-activated naõÈ ve splenocytes also produce TCA3 proteins but at lower levels than T cell clones (Luo et al., 1993).
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators TCA3 is expressed as an early cell activation gene. TCA3 or P500 mRNA can be detected as early as 1 hour after T cell activation with antigen or mitogen. mRNA accumulates to an apparent peak level by 4 hours in antigen-activated T cells then decreases in abundance (Wilson et al., 1988). These profiles
paralleled the IFN transcription pattern. In addition, anti-CD3-activated TCR , NK1.1 , CD4ÿ, CD8ÿ, NK-T cells make high levels of TCA3 mRNA (Kennedy et al., 2000). The signals that induce TCA3 and other cytokines can be dissociated among individual T cell clones (Kuchroo et al., 1993; Wilson et al., 1988). Furthermore, TCA3 transcription marks T cell activation even in the absence of a proliferative response (Wilson et al., 1988). TCA3 was not induced following IL-2 stimulation (Burd et al., 1987). Stimulation through the T cell receptor can be bypassed by simultaneous pharmacologic activation of protein kinase C and increase of intracellular calcium (Wilson et al., 1988). The data suggest that TCA3 is induced by activation through the TCR, but not by activation via the IL-2 receptor. Like many other inducible lymphokines, expression of TCA3 mRNA was completely blocked by cyclosporin A treatment (Burd et al., 1987). Mast cells or mast cell lines are induced to transcribe TCA3 in response to crosslinking of IgE receptors by IgE and specific multivalent antigens. In selected instances TCA3 expression can also be induced by activation of mast cells with Con A or phorbol ester plus ionophore (Burd et al., 1989). An alternative mast cell activation pathway capable of triggering TCA3 mRNA expression may also exist (Talkington and Nickell, 2001). Stimulation of mast cells with the growth factor IL-3, failed to induce TCA3 transcripts (Burd et al., 1989).
RECEPTOR UTILIZATION TCA3 binds with high (2 nM) affinity to the mouse chemokine receptor CCR8; I-309 partially inhibits this interaction (Goya et al., 1998). CCR8 is primarily expressed on a subset of thymocytes and on activated polarized TH2 cells (Kremer et al., 2001; Zingoni et al., 1998). Mouse macrophages, mouse mesangial cells, and rat vascular smooth muscle cells that do not express CCR8 also bind 125I-TCA3 with a similar Kd value (3±4 nM) (Luo et al., 1996; Luo and Dorf, 1996; Goya et al., 1998). Thus, one or more additional chemokine receptors can bind TCA3.
IN VITRO ACTIVITIES
In vitro findings TCA3 displays chemoattractant and activating activities on a variety of hematopoietic and nonhematopoietic 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
10ÿ11±10ÿ9 M
Devi et al., 1995
Chemoattractant
10ÿ9±10ÿ7 M
Luo et al., 1994
Nitrite production ÿ
O2 production
Macrophage/monocyte
Devi et al., 1995
ÿ7
M
Devi et al., 1995
ÿ8
ÿ7
M
Devi et al., 1995
10 ±10 10 ±10 10ÿ7 M
Cell adhesion
Devi et al., 1995
ÿ11
10
ÿ9
±10
Devi et al., 1995
ÿ7
M
Luo et al., 1994
ÿ8
ÿ7
M
Devi et al., 1995
10 ±10 10 ±10
Calcium mobilization
10ÿ8 M
Luo et al., 1994
ÿ11
ÿ8
M
Devi et al., 1995
ÿ12
ÿ7
M
Zingoni et al., 1998
10
±10
Chemoattractant
10
Anti-apoptotic
10ÿ10 M
Cell adhesion
M
ÿ8
Nitrite production
Chemoattractant Vascular smooth muscle
M
ÿ8
10 ±10
Granule exocytosis
Granule exocytosis
Mesangial cells
ÿ7
H2O2 production
Chemoattractant
T lymphocytes
ÿ8
±10
ÿ11
10
±10
ÿ9
10 ±10 ÿ8
Van Snick et al., 1996 ÿ8
ÿ7
M
M
Luo et al., 1994 Luo et al., 1994
Growth/survival
10
Cell adhesion
10ÿ11±10ÿ8 M
Luo et al., 1996
Chemotaxis
10ÿ10±10ÿ7 M
Luo et al., 1996
Growth/survival
ÿ8
10
M
M
ÿ10
Microglia
Chemoattractant
10
Astrocytes
Chemoattractant
10ÿ8 M
and activating capacities are coordinated. At the lowest concentrations of TCA3, when very few receptors are engaged, cell adhesion to matrix proteins increases and cells begin to migrate. As the target cells reach the focus of TCA3 production, where the TCA3 concentration should be the highest, phagocytic target cells become activated to degranulate and release proteolytic enzymes and other potentially tissue-destructive agents (Devi et al., 1995). In all cases tested TCA3-induced responses are sensitive to pertussis toxin treatment, indicating a critical role for G i protein-coupled receptors in TCA3-mediated signal transduction. TCA3 also induced anti-apoptotic activity affecting the survival and growth of some T lymphocytes and possibly non-hematopoietic cells (Table 1). In vitro P500 is a chemoattractant for macrophages, monocytic cell lines, and microglia (Laning, 1995). Intraperitoneal injection of P500 also induces a rapid inflammatory response in vivo characterized by a predominant monocytic infiltrate with little or
Luo et al., 1994
Luo et al., 1996 ÿ8
±10
M
Hayashi et al., 1995 Heesen et al., 1996
no lymphocyte or neutrophil involvement (Laning, 1995).
Bioassays used The most common and one of the most sensitive assays used for TCA3 is chemotaxis. The glycosylated and nonglycosylated forms of rTCA3 have similar chemotactic properties (Laning, 1995).
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS J558 myeloma cells were transfected with an expression vector for TCA3 to study the pathophysiological effects of TCA3 overproduction (Laning et al., 1994). For tumor transplantation, transfected
6
Martin E. Dorf
or control cells were injected subcutaneously into the flank of syngeneic Balb/c mice. The control and vector transfected tumors grew progressively at the same rate, but only 11% of tumors in the TCA3transfected group continued to progress (Laning et al., 1994). Some of the surviving recipients of the TCA3-transfected cells were challenged with nontransfected control J558 cells on the opposite flank; none of these mice developed a detectable tumor mass during the 23-day observation period, while all naõÈ ve recipients developed progressively growing tumor masses. Reciprocal experiments with a second TCA3-transfected Balb/c myeloma, P3X, were performed to demonstrate tumor specificity. TCA3transfected P3X cells provided protection to challenge with normal P3X cells, but not against J558. Similarly mice that recovered from the TCA3transfected J558 tumors were completely susceptible to P3X (Laning et al., 1994). The immunity was TCA3-dependent, as priming with irradiated tumor cells provided little protection against challenge with nontransfected tumor cells. Mixing TCA3transfected cells with normal tumor cells caused retarded growth of the normal tumor cells, provided the latter were injected into the same site. Furthermore, direct in situ injection of TCA3 early during the course of tumor implantation also inhibited tumor growth (Laning et al., 1994). Histologic examination of the TCA3-transfected tumor injection site identified infiltrating neutrophil and macrophage cell populations without noticeable accumulation of lymphocytes. The combined data suggest that TCA3 stimulates or augments tumor immunogenicity perhaps by attracting phagocytic cells that facilitate tumor destruction and presentation of tumor antigens to the immune system. Thus, TCA3 was viewed as a biological adjuvant (Laning et al., 1994). To test the concept that TCA3 could serve as an adjuvant for cell-mediated immunity, a TCA3 expression plasmid containing a muscle-specific promotor was co-injected into muscle along with an HIV-specific DNA vaccine. Histologic analysis of the injection site revealed predominance of infiltrating mononuclear cells with the TCA3 plasmid. In contrast, no accumulation of inflammatory cells was detected in controls (Tsuji et al., 1997). Addition of TCA3 also enhanced delayed-type hypersensitivity (DTH) and cytolytic T cell (CTL) responses (Tsuji et al., 1997). The effects were TCA3-specific as administration of anti-TCA3 suppressed DTH responses. Increases in HIV-specific IgG2a titers suggested that inoculation of the TCA3 plasmid skewed the response toward TH1 cells. The combined
results imply that TCA3 can act as an adjuvant by recruiting antigen-presenting cells and enhancing cell-mediated immunity.
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Role in experiments of nature and disease states Chemokines have been associated with the development of several cell-mediated autoimmune diseases. Expression of TCA3 transcripts were first correlated with the encephalitogenic potency of T cell clones (Kuchroo et al., 1993). These findings were supported by the observation that TCA3 mRNA was specifically induced in spinal cord 1±2 days before clinical signs of experimental allergic encephalomyelitis (EAE) were apparent (Godiska et al., 1995). Another study demonstrated the correlation of TCA3 upregulation with enhanced neutrophilia in EAE (Tran et al., 2000). The expression of CCR8, a TCA3 receptor, also correlates with the development of inflammatory lesions in EAE (Fischer et al., 2000). Administration of anti-TCA3 monoclonal antibody to cryptococcal immune mice directly demonstrated the role of TCA3 in protection against this fungal pathogen. Anti-TCA3 treatment also reduced TH1-mediated DTH responses to cryptococcal antigens and influenced the migration of leukocytes into the DTH reaction site (Doyle and Murphy, 1999). Anti-TCA3 treatments reduced both the number of neutrophils entering the DTH site and the amount of the T cell chemoattractant, MIP-1 , that the neutrophils produced (Doyle and Murphy, 1999). Thus, TCA3 was indirectly involved in recruiting lymphocytes into the DTH reaction. As EAE is also mediated by TH1 cells and is associated with expression of TCA3 and MIP-1 ; this chemokine network model may also apply to the development of cell-mediated autoimmune diseases.
ACKNOWLEDGEMENTS This work was partially supported by NIH grant CA 67416.
TCA3/Mouse CCL1 7
References Brown, K. D., Zurawski, S. M., Mosmann, T. R., and Zurawski, G. (1989). A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes. J. Immunol. 142, 679±687. Burd, P. R., Freeman, G. J., Wilson, S. D., Berman, M., Dekruyff, R., Billings, P. R., and Dorf, M. E. (1987). Cloning and characterization of a novel T cell activation gene. J. Immunol. 139, 3126±3131. Burd, P. R., Rollins, B. J., Wilson, S. D., Billings, P. R., Stiles, C. D., and Dorf, M. E. (1988). Comparison of fibroblast and T-cell activation genes. Cell. Immunol. 115, 481±483. Burd, P. R., Rogers, H. W., Gordon, J. R., Martin, C. A., Jayaraman, S., Wilson, S. D., Dvorak, A. M., Galli, S. J., and Dorf, M. E. (1989). Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J. Exp. Med. 170, 245±257. Devi, S., Laning, J., Luo, Y., and Dorf, M. E. (1995). Biologic activities of the beta-chemokine TCA3 on neutrophils and macrophages. J. Immunol. 154, 5376±5383. Doyle, H. A., and Murphy, J. W. (1999). Role of the C-C chemokine, TCA3, in the protective anticryptococcal cell-mediated immune response. J. Immunol. 162, 4824±4833. Fischer, F. R., Santambrogio, L., Luo, Y., Berman, M. A., Hancock, W. W., and Dorf, M. E. (2000). Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression. J. Neuroimmunol. 110, 195±208. Godiska, R., Chantry, D., Dietsch, G. N., and Gray, P. W. (1995). Chemokine expression in murine experimental allergic encephalomyelitis. J. Neuroimmunol. 58, 167±176. Goya, I., Gutierrez, J., Varona, R., Kremer, L., Zaballos, A., and Marquez, G. (1998). Identification of CCR8 as the specific receptor for the human beta-chemokine I-309: cloning and molecular characterization of murine CCR8 as the receptor for TCA-3. J. Immunol. 160, 1975±1981. Hayashi, M., Luo, Y., Laning, J., Strieter, R. M., and Dorf, M. E. (1995). Production and function of monocyte chemoattractant protein-1 and other beta-chemokines in murine glial cells. J. Neuroimmunol. 60, 143±150. Heesen, M., Tanabe, S., Berman, M. A., Yoshizawa, I., Luo, Y., Kim, R. J., Post, T. W., Gerard, C., and Dorf, M. E. (1996). Mouse astrocytes respond to the chemokines MCP-1 and KC, but reverse transcriptase-polymerase chain reaction does not detect mRNA for the KC or new MCP-1 receptor. J. Neurosci. Res. 45, 382±391. Keizer, D. W., Crump, M. P., Lee, T. W., Slupsky, C. M., ClarkLewis, I., and Sykes, B. D. (2000). Human CC chemokine I-309, structural consequences of the additional disulfide bond. Biochemistry 39, 6053±6059. Kennedy, J., Vicari, A. P., Saylor, V., Zurawski, S. M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Zlotnik, A. (2000). A molecular analysis of NKT cells: identification of a class-I restricted T cell-associated molecule (CRTAM). J. Leukoc. Biol. 67, 725±734. Kremer, L., Carramolino, L., Goya, I., Zaballos, A., Gutierrez, J., Moreno-Ortiz, M. D. C., Martinez, A. C., and Marquez, G. (2001). The transient expression of C-C chemokine receptor 8 in thymus identifies a thymocyte subset committed to become CD4 single-positive T cells. J. Immunol. 166, 218±225.
Kuchroo, V. K., Martin, C. A., Greer, J. M., Ju, S. T., Sobel, R. A., and Dorf, M. E. (1993). Cytokines and adhesion molecules contribute to the ability of myelin proteolipid protein-specific T cell clones to mediate experimental allergic encephalomyelitis. J. Immunol. 151, 4371±4382. Laning, J. C., (1995). Biochemical and functional characterization of the murine beta-chemokine, TCA3. PhD thesis, Harvard University, Cambridge, MA. Laning, J., Kawasaki, H., Tanaka, E., Luo, Y., and Dorf, M. E. (1994). Inhibition of in vivo tumor growth by the beta chemokine, TCA3. J. Immunol. 153, 4625±4635. Luo, Y., and Dorf, M. E. (1996). Beta-chemokine TCA3 binds to mesangial cells and induces adhesion, chemotaxis, and proliferation. J. Immunol. 156, 742±748. Luo, Y., Laning, J., and Dorf, M. E. (1993). Serologic analysis of a murine chemokine, TCA3. J. Immunol. 150, 971±979. Luo, Y., Laning, J., Devi, S., Mak, J., Schall, T. J., and Dorf, M. E. (1994). Biologic activities of the murine beta-chemokine TCA3. J. Immunol. 153, 4616±4624. Luo, Y., D'Amore, P. A., and Dorf, M. E. (1996). Beta-chemokine TCA3 binds to and activates rat vascular smooth muscle cells. J. Immunol. 157, 2143±2148. Miller, M. D., Hata, S., De Waal Malefyt, R., and Krangel, M. S. (1989). A novel polypeptide secreted by activated human T lymphocytes. J. Immunol. 143, 2907±2916. Miller, M. D., Wilson, S. D., Dorf, M. E., Seuanez, H. N., O'Brien, S. J., and Krangel, M. S. (1990). Sequence and chromosomal location of the I-309 gene. Relationship to genes encoding a family of inflammatory cytokines. J. Immunol. 145, 2737±2744. Natori, Y., Sekiguchi, M., and Ou, Z. (1997). Gene expression of CC chemokines in experimental crescentic glomerulonephritis (CGN). Clin. Exp. Immunol. 109, 143±148. Oh, C. K., and Metcalfe, D. D. (1994). Transcriptional regulation of the TCA3 gene in mast cells after Fc epsilon RI cross-linking. J. Immunol. 153, 325±332. Oh, C. K., Neurath, M., Cho, J. J., Semere, T., and Metcalfe, D. D. (1997). Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells. Biochem. J. 323, 511±519. Talkington, J., and Nickell, S. P. (2001). Role of Fc gamma receptors in triggering host cell activation and cytokine release by Borrelia burgdorferi. Infect. Immun. 69, 413±419. Teuscher, C., Butterfield, R. J., Ma, R. Z., Zachary, J. F., Doerge, R. W., and Blankenhorn, E. P. (1999). Sequence polymorphisms in the chemokines Scya1 (TCA-3), Scya2 (monocyte chemoattractant protein (MCP)-1), and Scya12 (MCP-5) are candidates for eae7, a locus controlling susceptibility to monophasic remitting/nonrelapsing experimental allergic encephalomyelitis. J. Immunol. 163, 2262±2266. Tran, E. H., Prince, E. N., and Owens, T. (2000). IFN-gamma shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164, 2759±2768. Tsuji, T., Fukushima, J., Hamajima, K., Ishii, N., Aoki, I., Bukawa, H., Ishigatsubo, Y., Tani, K., Okubo, T., Dorf, M. E., and Okuda, K. (1997). HIV-1-specific cell-mediated immunity is enhanced by co-inoculation of TCA3 expression plasmid with DNA vaccine. Immunology 90, 1±6. Van Snick, J., Houssiau, F., Proost, P., Van Damme, J., and Renauld, J. C. (1996). I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasoneinduced apoptosis. J. Immunol. 157, 2570±2576. Wilson, S. D., Burd, P. R., Billings, P. R., Martin, C. A., and Dorf, M. E. (1988). The expression and regulation of a potential lymphokine gene (TCA3) in CD4 and CD8 T cell clones. J. Immunol. 141, 1563±1570.
8
Martin E. Dorf
Wilson, S. D., Billings, P. R., D'eustachio, P., Fournier, R. E., Geissler, E., Lalley, P. A., Burd, P. R., Housman, D. E., Taylor, B. A., and Dorf, M. E. (1990a). Clustering of cytokine genes on mouse chromosome 11. J. Exp. Med. 171, 1301±1314. Wilson, S. D., Kuchroo, V. K., Israel, D. I., and Dorf, M. E. (1990b). Expression and characterization of TCA3: a murine inflammatory protein. J. Immunol. 145, 2745±2750. 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.
(1998). The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J. Immunol. 161, 547±551. Zlotnik, A., and Yoshie, O. (2000). Chemokines: a new classification system and their role in immunity. Immunity 12, 121±127.
LICENSED PRODUCTS 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
