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Trem-Like Transcript 2 Is Expressed on Cells of the Myeloid/Granuloid and B Lymphoid Lineage and Is Up-Regulated in Response to Inflammation

Department of Microbiology, Division of Developmental and Clinical Immunology, University of Alabama, Birmingham, AL 35294

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The triggering receptor expressed on myeloid cells (TREM) gene cluster encodes a group of transmembrane proteins that are emerging as important components in innate and adaptive immunity. In both mice and humans, the TREM gene cluster encodes eight receptors; only four of these, however, are direct homologs: TREM-1, TREM-2, TREM-like transcript 1 (TLT1), and TLT2. Of the transmembrane receptors encoded by the four conserved genes within this cluster, TLT2 has not been studied previously. Data presented in this study demonstrate that TLT2 is expressed early in B cell development in conjunction with B220 and is detected on all developing mouse B cell populations as well as B cells in the periphery. TLT2 expression on B cells in the periphery exhibits a distinct hierarchy with the highest detectable levels observed on B1 B cells in the peritoneum. The overall gradation of TLT2 expression on B cells is: B1 > marginal zone/transitional 2 > transitional 1 > follicular. Additionally, TLT2 expression was observed on mouse neutrophils throughout the body. Although monocytes were not observed to express TLT2, resident peritoneal and lung macrophages do express TLT2, suggesting that it is up-regulated in association with terminal differentiation of monocytes. Finally, both neutrophils and macrophages were observed to up-regulate TLT2 expression in vivo in response to inflammatory stimuli, whereas TLT2 expression on B cells remained unchanged. In conclusion, the data suggest that TLT2 may be involved in the innate immune response based on its expression profile and the fact that it is up-regulated in response to inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The innate immune system is not only responsible for pathogen recognition and clearance, but is also involved in the activation and regulation of the adaptive immune response. To accomplish this, the innate immune system relies on germline-encoded receptors, which have evolved to recognize invariant patterns associated with pathogens or endogenous danger signals that promote cellular activation. The triggering receptor expressed on myeloid cells (TREM)² cluster of genes encodes a group of transmembrane proteins that are emerging as important components involved in innate immunity. In both mice and humans, the TREM gene cluster encodes eight receptors; only four of these, however, are direct homologs: TREM-1, TREM-2, TREM-like transcript 1 (TLT1), and TLT2 (1). Most of the receptors encoded in the TREM gene cluster are activating receptors that associate with the transmembrane adaptor protein DAP-12, using the ITAM of this adaptor for function (1).

Of the conserved members in the TREM gene cluster, the best characterized is TREM-1. TREM-1 is expressed on neutrophils and a subset of monocytes and macrophages. Ligation of TREM-1 with anti-TREM-1 mAb results in the expression of cell surface activation markers and inflammatory cytokine production (2). Exposure to extracellular pathogens or the bacterial product LPS in vitro promotes up-regulation of TREM-1 expression on neutrophils and monocytes, and in vivo TREM-1 is strongly expressed at sites of inflammation (3). TREM-1 blockade using a soluble TREM-1:Fc fusion protein was observed to protect mice from LPS-induced septic shock and bacterial sepsis (3). Interestingly, alternative splicing of TREM-1 produces a soluble form of this receptor that has been shown to be secreted in response to inflammatory stimuli and to modulate the biological effects of inflammatory cytokines (4). These observations offer an elegant model for the regulation of TREM-1 signaling and provide physiological context for the receptor blockade studies. Thus, TREM-1 has emerged as not only an activating receptor, but as a central component in the process leading to amplification and/or maintenance of the inflammatory response.

Like TREM-1, the function of TREM-2 is dependent on its association with the adaptor molecule DAP-12. Unlike TREM-1, TREM-2 is not constitutively expressed on granulocytes or monocytes, and is expressed instead on immature monocyte-derived dendritic cells, osteoclasts, and microglia (5, 6, 7). In contrast to TREM-1, agonists that promote dendritic cell activation and maturation result in the down-regulation of TREM-2 expression (5). Signaling through TREM-2 has been shown to promote dendritic cell survival and the expression of the chemokine receptor CCR7 (5). Thus, it has been suggested that signaling through TREM-2 may be critical for dendritic cell maturation and migration to lymph nodes, thereby promoting Ag presentation to T cells. It has been reported that humans deficient in TREM-2 display symptoms of Nasu-Hakola disease (8, 9). Nasu-Hakola disease is marked by the presence of cystic bone lesions and loss of white matter in the brain, which lead to spontaneous bone fractures and presenile dementia, respectively. An identical disease phenotype is caused by a deficiency in DAP-12 expression, indicating that loss of TREM-2-mediated signal transduction via DAP-12 is responsible for the disease (8).

TLT1, unlike the other characterized TREM molecules, does not contain a charged residue within its transmembrane domain that would mediate the interaction of this receptor with DAP-12 (1). The cytoplasmic tail of TLT-1, however, contains an ITIM allowing for the interaction with Src homology region 2 domain-containing phosphatase 1 (SHP1), suggesting an inhibitory role for this receptor (10). Interestingly, TLT1 is expressed exclusively in megakaryocytes and platelets (10, 11). TLT1 is stored in the -granules of platelets and is quickly redistributed to their surface upon activation. It has also been reported that ligation of this receptor promotes rather than inhibits Ca²⁺ mobilization through an interaction with SHP2, although the physiological relevance of this, as well as the natural ligand responsible, have yet to be determined (12).

Of the conserved TREM gene cluster molecules, the last to be characterized is TLT2. Using mAbs raised against TLT2, we have analyzed its expression on murine hemopoietic cells. TLT2 is the only TREM molecule to be found on lymphocytes. TLT2 is expressed early in B cell development, appearing on the cell surface in concert with the B lineage marker B220. Within the B cell compartment, TLT2 expression is higher on marginal zone (MZ) and transitional subsets than on follicular (FO) B cells, and the highest level of expression is observed on B1 B cells. Although monocytes in the bone marrow or peripheral blood do not express detectable levels of TLT2, its expression is up-regulated in conjunction with differentiation into macrophages. Finally, TLT2 is present on neutrophils in the bone marrow as well as the periphery, and inflammatory stimuli result in a dramatic increase in the expression of TLT2 on these cells in vivo.

	Materials and Methods

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Cloning of TLT2

The human tlt2 gene was amplified from cDNA generated from the human Daudi B cell line using primers: 5'-CCTTGGATCTCCAGGCCTGACACTGCC and 3'-TCAGATCAAGTAGACTTCCACATAGGG. The sequence of the human tlt2 gene was identical with the sequence provided in GenBank. At the time, the complete mouse gene sequence was not available in the public database. To identify the mouse tlt2 gene, a reverse blast search was conducted of mouse expressed sequence tag (EST) libraries using the human TLT2 sequence. This search revealed seven ESTs displaying homology to the Ig region of the human TLT2 protein with accession numbers: AV140744, AV142748, AV143551, AV145466, AV146020, BB611200, and BB630804. The consensus sequence derived from these ESTs was used to generate primers for mouse tlt2, which were used to obtain the complete sequence of mouse tlt2 using RACE-PCR. RACE-PCR was conducted on RACE ready cDNA generated from the K46 murine B cell lymphoma using the Smart RACE system (BD Clontech) with the primers: 5'-GCCGTGTACGCATACACCAGTATCGTCCG and 3'-CTGCTGTCCAACCCTGGGAGGTCAGG. The full-length transcript for mouse tlt2, including the 5' and 3' untranslated regions, was subsequently amplified using primers: 5'-GACTGTGAGCAGGAGTGACTGAAG and 3'-GTCGCACATGTTTATGTCATCCATTGCATCC. The GenBank accession number for the mouse tlt2 nucleotide sequence is DQ341272 (www.ncbi.nlm.nih.gov/GenBank/index.html).

RT-PCR

RNA was isolated using the RNAeasy isolation kit and protocols (Qiagen). For RT-PCR, cDNA was generated using the Thermoscript II system (Invitrogen Life Technologies). For RACE-PCR, cDNA was generated using the polymerase and primers provided with the Smart RACE system (BD Clontech). All PCR were performed using the KOD high fidelity polymerase system (Novagen).

Ab production

The extracellular region of mouse TLT2 (mTLT2) was expressed as a fusion with the Fc portion of human IgG1. The hinge, CH2, and CH3 portion of the human IgG1 gene was cloned into the pShuttle CMV expression vector (Qbiogene). The extracellular region of mTLT2 was then cloned into the resulting vector. This construct was used to generate recombinant adenovirus expressing a soluble mTLT2-Fc fusion protein using the AdEasy system (Qbiogene). The recombinant virus was used to infect the HEK293 cell line, resulting in the secretion of mTLT2-Fc into the culture medium. Purification of the TLT2-Fc recombinant protein was performed using protein A chromatography following protocols for purification of mouse Abs that involve the removal of bovine Ig.

To generate Abs against mTLT2, Sprague-Dawley rats 10 wk of age were immunized in a hind footpad with 100 µg of rat TLT2-Fc protein in 50 µl of PBS mixed with 50 µl of Titermax adjuvant (Sigma-Aldrich). Four injections were performed: once every 2 wk, followed by a final immunization without adjuvant 1 wk after this series. The following day, lymphocytes from the popliteal lymph node of the immunized limb were harvested and fused to the Ag8.653 fusion partner (American Type Culture Collection) following standard protocols.

Screening of the resulting hybridomas was performed by staining cells expressing mTLT2 fused to GFP in place of the cytoplasmic domain. The extracellular and transmembrane regions of mTLT2 were cloned into pGFPN1 (BD Clontech). The 293T cells were transiently transfected with this construct and stained with supernatants harvested from hybridoma clones. The samples were then stained with a secondary anti-rat Ig Ab conjugated to PE, and the samples were analyzed by flow cytometry. Hybridomas displaying specific reactivity against the TLT2-GFP-positive cells were isolated and expanded.

Cell culture

The HEK293 cell line was cultured in DMEM medium supplemented with 10% FBS (HyClone), 2 µM L-glutamine, 50 µM 2-ME, 100 µg/ml streptomycin-penicillin, and 50 µg/ml gentamicin (Sigma-Aldrich) at 37°C under 5% CO₂. Hybridomas were expanded in RPMI 1640 complete medium supplemented with Hybridoma Cloning and Fusion Supplement (Roche). After expansion, hybridomas were cultured in complete RPMI 1640 supplemented with 5% ultra low FBS (Invitrogen Life Technologies). Transient transfection of HEK293 cells was conducted using LipofectAMINE 2000 following the manufacturer’s suggested protocols (Invitrogen Life Technologies).

Cell isolation

C57BL/6 mice (The Jackson Laboratory) 8–10 wk of age were used for isolation of tissues and cells. All mice were housed in specific pathogen-free conditions in University of Alabama at Birmingham animal facilities, and all procedures were approved by an institutional review committee. Peritoneal cells were isolated by peritoneal lavage with 7 ml of HBSS. Peripheral blood was isolated by cardiac puncture. Splenocytes were isolated by gentle dissociation of the spleen using frosted glass slides. Bone marrow cells were isolated by flushing femurs with HBSS. RBCs were lysed by incubating cells in RBC lysis buffer (10 mM KHCO₃, 150 mM NH₄Cl, and 0.1 mM EDTA) for 3 min on ice.

For experiments involving elicited macrophages, mice were injected i.p. with 1 ml of thioglycolate broth (BCI). Seventy-two hours after i.p. injection, mice were sacrificed and peritoneal cells were harvested, as described above.

For studies involving experimentally induced inflammatory responses, mice were injected i.p. with 50 µg or i.v. with 25 µg of LPS serotype 0111:B4 or 25 µg of staphylococcal enterotoxin B (SEB) (Sigma-Aldrich).

For B cell stimulation experiments, splenocyte preparations were enriched for B cells by Thy-1-mediated complement depletion of T cells. Complement-mediated T cell depletion was performed, as described, using hybridoma supernatants from clones HO13 and T24, followed by incubation for 40 min at 37°C with low toxicity rabbit complement (Cedarlane Laboratories).

Abs and reagents

For flow cytometry, anti-B220 PerCP/allophycocyanin, CD5 PerCP/FITC, CD11b FITC/allophycocyanin, Gr1 allophycocyanin, CD11c FITC, CD21 FITC, and CD23 FITC were purchased from BD Biosciences. Anti-IgM FITC/allophycocyanin, anti-IgD FITC, and streptavidin-PE/allophycocyanin were purchased form Southern Biotechnology Associates. Abs to mTLT2 1H4 and 1C5 were biotinylated using EZ-Link succinimidyl-2-(biotinamide)-ethyl-1,3'-dithiopropionate biotin (Pierce).

Flow cytometry

Cells (1 x 10⁶/sample) were washed twice in FACS buffer (PBS, 0.01% NaN₃⁺, and 0.5% FBS) and then incubated with the appropriate Ab mixture in 96-well microtiter plates for 30 min on ice. The Ab-labeling mixture contained l µg/sample of 24G2 mAb (BD Biosciences) to block FcR binding. After this incubation, the cells were washed twice in FACS buffer. When necessary, the samples were incubated with secondary reagents for an additional 30 min, followed by two additional washes in FACS buffer. Samples were either analyzed immediately or resuspended in 2% paraformaldehyde. All steps were performed on ice. Samples were run on either a FACScan or FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest or WinMidi software.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cloning of TLT2

A search of human public EST databases was conducted to identify genes of unknown function that are expressed in lymphocytes. Based on the results of this screen, tlt2, a gene encoding a transmembrane protein resembling the natural cytotoxicity receptor NKp44, was chosen as a gene of interest. EST expression data suggested that TLT2 is expressed in B cells, as over half of the ESTs listed were derived from either a lymphoid organ or purified B cell sources. Primers generated to screen for the expression of the human tlt2 transcript revealed the presence of tlt2 mRNA in cell lines representing pro-, pre-, and mature B cell subsets in humans (data not shown). The tlt2 transcript was not detected, however, in cell lines representing human T lymphocytes or monocytes (data not shown). These data were consistent with the expression profile generated from EST entries in the database.

Using the predicted sequence for human tlt2, a tblastn search was conducted of murine EST databases. This search generated several ESTs encoding proteins with homology to the Ig region of human TLT2. The consensus sequence of these ESTs was used to generate primers to amplify a region of the putative mouse homolog of the human tlt2 gene. These primers were used to probe for the tlt2 transcript, and the expression of this transcript was detected in cDNA derived from murine B cell lines as well as total splenic cDNA (data not shown). Following this initial screen, cDNA derived from the murine B lymphoma cell line K46 was used as a source for RACE-PCR. The resulting 2704-bp transcript encoding the mouse homolog of the human tlt2 gene was subsequently amplified from total splenic cDNA (Fig. 1). Comparison of the mouse tlt2 transcript with the mouse genome revealed that mouse tlt2 is encoded by six exons spanning 11 kb of mouse chromosome 17C within the TREM gene cluster.

View larger version (106K): [in this window] [in a new window]   FIGURE 1. Nucleic and amino acid sequence of mTLT2. The full-length nucleotide sequence encoding TLT2 was obtained by RACE-PCR with primers based on a consensus sequence derived from a tblastn search of mouse EST databases using the amino acid sequence of human TLT2. The GenBank accession number for the mouse tlt2 nucleotide sequence is DQ341272. Amino acids in the open reading frame are represented by one-letter symbols.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. Alignment of amino acid sequences for human TLT2 and mTLT2. Comparison of human TLT2 and mTLT2 at the amino acid level reveals that the proteins are 50% identical and 60% homologous. Identical amino acids are denoted by asterisks. The N-terminal leader sequence is represented by a line over the amino acid sequences. The variable type Ig domain is denoted by the boxed-in region. The serine threonine-rich region within the extracellular domain in marked by a dashed arrow, and the transmembrane domain is underlined.

To determine the expression pattern for TLT2 in the mouse, a panel of mAbs was generated against the extracellular domain of mTLT2. The mAbs were screened against transfected HEK293 cells that expressed a TLT2:GFP fusion protein, and clone 1C5 was selected for analysis of TLT2 expression in hemopoietic cells based on the observation that the intensity of staining with 1C5 was directly proportional to the relative expression of TLT2:GFP by HEK293 cells (Fig. 3A). To further validate the specificity of anti-TLT2 mAb clone 1C5, bone marrow cells were isolated and stained with purified mAb preincubated in the presence (open histogram) or absence (solid histogram) of a 10-fold excess of the soluble TLT2:Fc recombinant protein. As can be seen in Fig. 3A, the soluble TLT2-Fc recombinant protein effectively blocked staining of bone marrow cells, demonstrating that clone 1C5 is specific for TLT2 and does not cross-react with other TREM gene cluster receptors. Using this reagent, we analyzed the expression of TLT2 on hemopoietic cells isolated from a variety of immunological organs. Single channel histograms depicting TLT2 staining of hemopoietic cells revealed that a large percentage of the cells in the spleen, lymph nodes, blood, bone marrow, and peritoneal cavity, 55, 30, 35, 69, and 71% of cells, respectively, express TLT2 (Fig. 3B). In contrast, TLT2 expression was undetectable on cells of thymic origin. The 1C5 anti-TLT2 mAb was next used in combination with Abs specific for additional cell surface markers to further characterize the TLT2⁺ cell types within the different anatomical locations. Within the lymphocyte population, cells exhibiting a small forward and side scatter profile, it was determined that TLT2⁺ cells were predominantly B220⁺ (Fig. 3C). In contrast, splenocytes stained with the T lymphocyte markers CD3, CD4, CD8, or the TCR were uniformly TLT2 negative (data not shown). Thus, it is apparent from the results that within the lymphoid lineage, TLT2 expression is restricted to B lymphocytes. These results were corroborated using a second anti-TLT2 mAb, clone 1H4, as are all of the results depicted in the subsequent figures (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. TLT2 is expressed on hemopoietic cells throughout the body. A, Analysis of anti-TLT2 mAb specificity. HEK293 cells were transfected with pGFPN1 encoding TLT2-GFP. After 24 h, transfected cells were incubated with anti-TLT2 mAb (clone 1C5), washed, and stained with a secondary anti-rat polyclonal Ab conjugated to PE (left panel). Bone marrow cells were isolated and incubated in the presence of anti-TLT2 mAb (1C5, 10 µg/ml, solid histogram) or in the presence of anti-TLT2 mAb that had been preincubated with a 10-fold excess of soluble rat TLT2:Fc protein for 30 min on ice (open histogram). Cells were then washed and stained with a secondary anti-rat polyclonal Ab conjugated to PE (right panel). B, Analysis of TLT2 expression on hemopoietic cells. Single-cell suspensions were prepared from a number of immunological organs, and the cells were stained with mAb specific for TLT2 (1C5). Significant expression of TLT2 was observed on all cell populations examined, with the exception of thymocytes. Expression of TLT2 was highest on cells isolated from the bone marrow and peritoneal cavity. The results are representative of a minimum of five independent experiments. C, Analysis of TLT2 expression on lymphocyte populations. Single-cell suspensions were prepared from the sites indicated, and the cells were stained with anti-B220 and anti-TLT2 mAbs. The lymphocyte population represented by cells with a small forward and side scatter profile was gated on revealing that B220+ cells are predominantly TLT2+, whereas B220– cells representing T lymphocytes are TLT2–. Additional flow cytometric analyses using T cell-specific markers (anti-CD4, -CD8, -CD3, and -/ TCR) were performed revealing that T cells are indeed TLT2 negative (data not shown).

As the site of hemopoiesis in the adult, the bone marrow contains populations of both mature recirculating B lymphocytes as well as a continuum of developing B cells. These developing B cells have been classified into distinct populations based on surface marker expression and critical checkpoints in their development. The earliest stage of progenitor commitment to the B cell lineage correlates with the expression of B220 on their cell surface. In the Hardy scheme of B cell development, fractions A-D express B220, but do not express IgM or IgD (13). Cells that express IgM, but not IgD, comprise fraction E, the immature B cell population, and mature B lymphocytes express both IgM and IgD.

To determine at what point during their development B lymphocytes express TLT2, we analyzed the expression of TLT2 on these subsets. By discriminating between IgD⁺ and IgD^– cells, we could compare the expression of TLT2 on fractions A-E, developing B cells, with that of fraction F, mature B cells. Whereas all B220⁺ cells express TLT2, its expression is slightly higher on developing cells compared with mature B cells (Fig. 4). This finding is consistent with the data below describing higher expression of TLT2 on transitional cells within the spleen compared with mature FO B cells (Fig. 5). Moreover, the expression of TLT2 within the heterogeneous population of developing B cells is uniform when comparing cells that express IgM on their surface (immature) vs those that do not (pro-B and pre-B). Thus, TLT2 is expressed in concert with the B lineage marker B220 at an early stage of development before expression of the BCR.

View larger version (64K): [in this window] [in a new window]   FIGURE 4. TLT2 is expressed throughout B cell development in the bone marrow. Single-cell suspensions were prepared and stained with mAbs to distinguish developing B cell populations within the bone marrow (B220+,IgD– cells, fractions (Fr.) A-E) from mature recirculating B cells (B220+,IgD+, fraction F) (upper left). Fractions A-E were further separated into pro-/pre-B cells (fractions A-D) and immature B cells (fraction E) based on the expression of IgM (lower left). Fractions A-D, E, and F were analyzed to determine the expression of TLT2, demonstrating that TLT2 is expressed early in B cell development in conjunction with B220 and that the level of expression remains constant (upper and lower right). TLT2 expression was consistently observed to be higher on developing B cells (fractions A-E) than on recirculating B cells (fraction F) (upper right). The data are representative of four independent experiments.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. TLT2 exhibits a gradient of expression on B lymphocyte subpopulations in the spleen. Flow cytometric analysis of B220+ splenocytes revealed that the relative level of TLT2 expression on B cells correlates directly with IgM expression and is inversely correlated with IgD expression (upper panels). B cell subsets in the spleen were further discriminated based on their expression of B220, IgM, and CD21 to identify T1 and T2, MZ, and FO B cell subpopulations (middle panels). These populations were then analyzed for TLT2 expression using anti-TLT2 mAb, revealing a gradient of TLT2 expression: T2/MZ > T1 > Fo (bottom panel). The data are representative of four independent experiments.

The peripheral B lymphocyte compartment is not a homogeneous population of cells; rather it is comprised of functionally and phenotypically distinct subpopulations (13). The most notable distinction with respect to the B cell compartment relates to functional/phenotypic differences between the B1 and B2 class of B lymphocytes. These two populations of B cells differ in Ag receptor gene usage, cell surface marker expression, and anatomical location. Within the B2 population, there exist functionally distinct subsets of B cells, which can be subdivided into two distinct transitional populations, transitional 1 (T1) and T2 cells, as well as FO and MZ based on differential expression of cell surface markers (13).

Based on flow cytometric analysis, it was determined that B lymphocytes present in the spleen and bone marrow that are phenotypically TLT2^high, are IgM^high and IgD^low (Fig. 5). The IgM^high and IgD^low cell surface phenotype on splenic B cells is generally indicative of either transitional or MZ populations. Consistent with this hypothesis, TLT2 expression is higher in the spleen than in the lymph nodes, which contain no MZ cells and from which transitional populations are excluded (Fig. 3). B lymphocytes in the spleen can be identified as T1, T2, MZ, or FO B cells based on the expression of the B cell marker B220, membrane IgM, and the low affinity IgE receptor CD21 (14). When the relative expression of TLT2 within these groups was analyzed, it was determined that T2/MZ cells exhibit the highest levels of TLT2 on their surface. T1 B cells express less TLT2 than do the T2/MZ populations, but more than FO B cells (Fig. 5). Thus, the hierarchy of TLT2 expression correlates with that of cell surface IgM expression, T2/MZ > T1 > FO.

B cells in the peritoneal cavity also differed in their expression of TLT2. Although the peritoneal cavity is the primary location of B1 cells, it contains B2 lymphocytes as well. B1 cells express the T cell Ag CD5 as well as the CD11b integrin on their surface, two features that readily distinguish them from their B2 counterparts. The B1 B cell population can be further subdivided into B1a (CD5⁺,CD11b⁺) and B1b cells (CD5^–,CD11b⁺) (13). When the expression of TLT2 was analyzed on cells within the peritoneal cavity, it was determined that B1a and B1b B cells express comparable levels of TLT2 that are considerably higher than that observed on B2 cells within the peritoneal cavity (Fig. 6).

View larger version (42K): [in this window] [in a new window]   FIGURE 6. TLT2 is expressed at higher levels on B1 vs B2 cells in the peritoneal cavity. Cells isolated by peritoneal lavage were stained to detect B220 and CD11b expression to distinguish B1a/b B cells from B2 B cells (upper left). The B220low/–,CD11blow population representing B1a and B1b B cells was analyzed for TLT2 and CD5 expression, revealing that CD5+,CD11blow B1a B cells express comparable levels of TLT2 when compared with CD5–,CD11blow B1b B cells (upper right). Comparison of TLT2 expression on B2 B cells from the peritoneal cavity vs B1a/b B cells revealed that TLT2 is expressed at significantly higher levels on the latter population.

Previous studies to characterize expression of proteins encoded within the TREM gene cluster have demonstrated their expression on cells of the myeloid lineage (7). Therefore, experiments were performed to determine whether TLT2 is expressed on monocytes or macrophages. Analysis of bone marrow demonstrated that the B220^–, Gr1^–, CD11b⁺ population representing developing and mature monocytes was negative for TLT2 expression (Fig. 7A). Similarly, analysis of circulating monocytes in the blood revealed that they too are negative for TLT2 expression (Fig. 7A). In contrast, resident macrophages isolated from the lung or peritoneal cavity exhibit significant levels of TLT2 expression, as depicted in Fig. 7, B and C. These data support the conclusion that TLT2 expression is up-regulated in conjunction with emigration of monocytes from the blood into tissues, where they differentiate into resident tissue macrophages.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. TLT2 expression is up-regulated in conjunction with differentiation of monocytes into resident tissue macrophages. A, Single-cell suspensions isolated from the bone marrow and blood were stained with mAbs to detect CD11b and Gr1 expression. The small forward/side scatter population of cells was analyzed, and the CD11b+,Gr1– population representing monocytes was analyzed for TLT2 expression. The data reveal that monocytes in the bone marrow and blood do not express TLT2. B, Analysis of TLT2 expression on resident alveolar macrophages. A single-cell suspension was prepared by lung lavage and the resident alveolar macrophage population identified based on forward scatter characteristics and the expression of CD11c and Gr1. The CD11c+,Gr1– resident macrophage population was analyzed for TLT2 expression, demonstrating that the cells are uniformly positive for TLT2 expression. C, Analysis of TLT2 expression on resident peritoneal macrophages. A single-cell suspension was prepared by peritoneal lavage, and the resident macrophage population was discriminated based on staining for B220, CD11b, and Gr1. The B220–,CD11b+,Gr1– resident macrophage population was then analyzed for TLT2 expression, revealing that all of the cells are positive.

In addition to the TLT2⁺,B220⁺ B cell and TLT2⁺,CD11b⁺,Gr1^– macrophage populations described above, it was noted that cells exhibiting a side scatter profile larger than that of lymphocytes or monocytes/macrophages also express TLT2. Although this population is present in the spleen and peripheral blood, it was observed at a considerably greater frequency in the bone marrow. When stained with Abs against the cell surface markers CD11b and Gr1, these cells in the bone marrow and peripheral blood were positive for both, identifying them as neutrophils (Fig. 8). As can be seen in Fig. 8, neutrophils isolated from either site are uniformly positive for TLT2 expression. Analysis of Gr1⁺,CD11b⁺ neutrophils in the spleen revealed that they also express TLT2, whereas cells expressing the dendritic cell marker CD11c are negative for TLT2 (data not shown).

View larger version (44K): [in this window] [in a new window]   FIGURE 8. TLT2 is uniformly expressed on neutrophils. Single-cell suspensions were prepared from the bone marrow and blood and stained with mAbs against CD11b and Gr1. The high side scatter population in bone marrow and blood was gated on (R2, upper panels) and analyzed for Gr1 expression. The Gr1+ cells (middle panels) were then gated on and analyzed for CD11b expression in addition to TLT2. As shown, Gr1+,CD11b+ neutrophils in the bone marrow and blood are uniformly positive for TLT2 expression (lower panels).

Previous studies have demonstrated that the expression of TREM gene cluster receptors, including TREM-1, TREM-2, and TLT1, is altered in response to cellular activation. To determine whether cellular activation alters TLT2 expression, experiments were performed both in vitro as well as in vivo to monitor TLT2 expression on B cells, macrophages, and neutrophils in response to activating stimuli. The expression of TLT2 is unique among the TREM molecules in that it is the only TREM receptor expressed on lymphocytes. To determine whether B cells alter their expression of TLT2 in response to activating stimuli, splenocytes were enriched for B lymphocytes by complement-mediated depletion of T cells. B cells were then incubated in the presence of various stimuli individually or in combination, including polyclonal anti-IgM F(ab')₂ Ab, anti-CD40 mAb (1C10), LPS, CpG DNA, PMA, or ionomycin; however, none of the stimuli tested altered the expression of TLT2 in vitro (data not shown). Given that the expression of TLT2 within the B cell compartment is highest on the B1 and MZ populations, which are predominantly responsible for the rapid recognition of bacterial pathogens in vivo, experiments were performed to determine whether exposure to the bacterial product LPS results in modulation of TLT2 expression on B cells in vivo. As depicted in Fig. 9, administration of LPS caused potent B cell activation based on up-regulation of the activation marker CD69. However, as was the case with in vitro stimulation, there was no change in TLT2 expression within the B cell compartment in response to endotoxin exposure. To further examine this, TLT2 expression on MZ B cells from the spleen and B1 B cells from the peritoneum was monitored in response to administration of LPS. Once again, there was no detectable increase in TLT2 expression on these B cell subpopulations, although there was a consistent, albeit minor decrease in TLT2 expression observed on MZ B cells isolated from mice injected with LPS when compared with cells from control animals. In summary, it appears that expression of TLT2 on B cells is not altered significantly in response to activation either in vitro or in vivo.

View larger version (44K): [in this window] [in a new window]   FIGURE 9. TLT2 expression on B cells does not increase in response to inflammation associated with LPS administration in vivo. C57BL/6 mice were injected with LPS either i.p. or i.v., and B cells from spleen or peritoneal cavity were isolated 24 h later. A, TLT2 is not up-regulated on splenic B cells in response to LPS. B cell activation was confirmed based on the up-regulation of CD69 on splenic B cells in response to LPS administration. In contrast, no detectable change in the level of TLT2 expression was observed on splenic B cells, regardless of whether LPS had been administered via the i.p. or i.v. route. B, TLT2 expression is not selectively up-regulated on MZ B cells in the spleen. Total splenic B cells were isolated from control and LPS-treated mice and stained with mAbs against B220, IgM, and CD21 to identify the T2/MZ population. LPS treatment was not observed to up-regulate TLT2 expression on this population, despite the fact that MZ cells exhibit more rapid and potent responses to LPS. C, TLT2 expression is not selectively up-regulated on B1 B cells in response to LPS. B1 B cells isolated from control and LPS-treated mice were analyzed for TLT2 expression. As seen, the data demonstrate that LPS does not promote increased expression of TLT2 on this subpopulation of B cells.

View larger version (64K): [in this window] [in a new window]   FIGURE 10. Neutrophils up-regulate TLT2 expression in response to inflammatory stimuli. C57BL/6 mice were sham injected with PBS or were injected with LPS or SEB via the i.p. or i.v. route. After 24 h, cells were isolated from the peritoneal cavity or peripheral blood and stained for Gr1, CD11b, and TLT2. The population of cells exhibiting high side scatter and Gr1 expression, representing neutrophils (left columns), was analyzed for CD11b and TLT2 expression. The relative level of TLT2 vs CD11b expression on neutrophils is depicted in the right columns. The mean fluorescence intensity for TLT2 expression on neutrophils isolated from control mice is indicated in each of the cytograms by a horizontal line as a reference point. The mean fluorescence intensity for TLT2 expression is indicated to the right of each TLT2 x CD11b cytogram. The data are representative of three independent experiments.

View larger version (59K): [in this window] [in a new window]   FIGURE 11. Macrophages up-regulate TLT2 expression in response to inflammation. C57BL/6 mice were left untreated or were injected i.p. with thioglycolate broth. Seventy-two hours later, cells were isolated from the peritoneal cavity by peritoneal lavage. The single-cell suspensions were stained with mAbs against B220, CD11b, and TLT2. Thioglycolate treatment was observed to cause a large influx of monocytes into the peritoneal cavity, where they underwent differentiation and priming, as shown by the increase in B220–, CD11bhigh cells (upper panels). Analysis of resident vs elicited macrophage populations for TLT2 expression revealed a 3- to 4-fold increase in the level of TLT2 expression on elicited macrophages (lower panel).

	Discussion

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Studies concerning the function of receptors encoded within the TREM gene cluster have revealed that members of this cluster are indeed involved in cellular activation and play a role in innate as well as adaptive immunity (7). TREM-1 is expressed on monocytes and neutrophils, and ligation of this receptor has been shown to promote the production of proinflammatory cytokines and chemokines (2, 3). Moreover, TREM-1 has been shown to amplify inflammatory responses in conjunction with signaling via TLRs. TREM-2 is expressed on immature monocyte-derived dendritic cells, osteoclasts, and microglial cells. Ligation of TREM-2 has been shown to play a role in activation and migration of dendritic cells (5). Additionally, TREM-2 plays an important role in brain function and bone remodeling (6, 8, 9). A common feature of the TREM receptors is the presence of a charged lysine residue in the transmembrane domain that mediates the noncovalent interaction with the DAP-12 transmembrane homodimer that contains ITAMs within its cytoplasmic domain (7). By virtue of the interaction with DAP-12, TREM molecules, as well as the related NKp44 receptor, are able to initiate tyrosine-based signaling leading to cellular activation (1, 2, 22). Other receptors encoded within the TREM gene cluster such as TLT1 lack the conserved transmembrane lysine residue, but contain within their own cytoplasmic domain tyrosine-based signaling motifs (11). In the case of TLT1, there is an ITIM, which mediates recruitment of SHP1 and SHP2 (11, 12).

TLT2 is unique in that it lacks either the conserved transmembrane lysine residue or ITAM/ITIMs within its own cytoplasmic domain. Thus, TLT2 does not exhibit any of the features associated with classical tyrosine-based signaling. This raises the question of whether TLT2 transduces a signal in response to ligand binding and, if so, how. Indeed, there is relatively little conservation between mouse and human TLT2 with regard to the amino acids contained in their respective cytoplasmic domains. Although a previous report suggested that the cytoplasmic domain of human TLT2 contains a YxxV motif that may play a role in endocytosis as well as recruitment of STAT3 and a second motif YxxC that might also recruit STAT3, comparison of the human and mouse sequences for TLT2 reveals that neither motif is conserved (1). Nevertheless, it has also been suggested that the cytoplasmic domain of human TLT2 contains a potential type I SH3-binding motif with the consensus sequence +xxPxxP, in which the + represents positively charged arginine (1). Alignment of mouse and human TLT2 reveals that there is an analogous +xPxxP sequence in the mouse. Thus, TLT2 may interact with one or more SH3 or WW domain-containing effector proteins either constitutively or in response to ligand binding. Studies have been performed using anti-TLT2 mAb in an attempt to determine whether TLT2 functions as an activating receptor in macrophages and B cells. Stimulation of peritoneal macrophages with soluble or plate-bound anti-TLT2 mAb was not observed to induce secretion of proinflammatory cytokines such as IL-1 or TNF- nor was anti-TLT2 mAb observed to potentiate proinflammatory cytokine production in response to LPS (data not shown). Stimulation of splenic B cells with anti-TLT2 mAb in soluble or plate-bound form was not observed to potentiate survival, induce proliferation, or up-regulate expression of activation markers, including CD69 and CD86. Moreover, anti-TLT2 mAb was not observed to potentiate or to attenuate any of these functional responses when added to B cell cultures in the presence of polyclonal anti-IgM (Fab')₂ Ab (data not shown). These results must be viewed with some degree of caution, however, as it is possible that the mAb used (clone 1C5) may not mimic binding of the natural ligand(s) for TLT2 and therefore may not induce the physiological response associated with ligand binding.

Although it remains to be determined whether TLT2 functions as an activating receptor similar to TREM-1 and TREM-2, data presented in this study support the conclusion that TLT2 may be important during the innate immune response triggered by inflammatory stimuli. TLT2 expression is increased on neutrophils in response to in vivo administration of LPS and SEB. A consistent finding related to this is the fact that increased expression of TLT2 on neutrophils directly correlates with up-regulation of CD11b, a known activation marker (15). In the case of LPS stimulation, it is possible that up-regulation of TLT2 is a direct consequence of TLR4-mediated signal transduction. Alternatively, it is equally possible that LPS indirectly affects TLT2 expression through the production of inflammatory cytokines such as IL-1 and TNF-. In support of the latter possibility, i.p. administration of SEB was observed to promote an influx of neutrophils within 24 h and these cells exhibited significant up-regulation of TLT2 in concert with CD11b. In vivo administration of SEB promotes the production of proinflammatory cytokines through its ability to bind to T cells that express specific TCR V subunits, or by binding to MHC class II on APCs (16, 17, 18, 19). It is formally possible that the inflammatory response caused by administration of SEB could induce MHC class II expression on neutrophils, thereby promoting direct activation of neutrophils through binding of SEB to MHC class II. However, it is more likely that SEB induces up-regulation of TLT2 on neutrophils via an indirect process that involves the production of proinflammatory cytokines, which in turn activate the neutrophils. Nevertheless, it remains to be determined whether TLT2 expression is modulated in response to the production of proinflammatory cytokines, or by direct stimulation of cells via activating receptors (e.g., TLRs), or both.

That TLT2 is involved in the inflammatory response is further supported by experiments demonstrating that thioglycolate-elicited macrophages exhibit increased TLT2 expression when compared with resident macrophages. Thus, inflammatory stimuli that promote recruitment and priming of macrophages induce expression of TLT2 as well. In contrast to neutrophils and macrophages, which increase TLT2 expression in response to inflammatory stimuli, B cells do not. In vivo administration of LPS, which is a potent B cell agonist that promotes polyclonal B cell activation, was not observed to alter TLT2 expression despite the fact that B cells were activated based on analysis of CD69 expression. Indeed, incubation of splenic B cells in vitro with a range of stimuli was not observed to induce up-regulation of TLT2. Thus, it remains to be determined whether B cells actually up-regulate TLT2 expression under any condition regardless of whether the response is associated with inflammation or the adaptive immune response.

In conclusion, this study demonstrates that TLT2 is expressed on cells of the innate immune system, including neutrophils and macrophages. Additionally, it was shown that expression of TLT2 is up-regulated on neutrophils and macrophages in response to inflammatory stimuli. Taken together, these findings suggest that TLT2 may play a role in the innate immune response. Moreover, the fact that TLT2 is expressed on cells of the B lineage, with the highest level of expression found on B1 and MZ B cells, raises the possibility that TLT2 is involved in the initial humoral immune response to bacterial pathogens and the development of autoimmune disease associated with these B cell subpopulations.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Louis B. Justement, Department of Microbiology, Division of Developmental and Clinical Immunology, 389 Wallace Tumor Institute, 1824 6th Avenue South, Birmingham, AL 35294-3300. E-mail address: lbjust{at}uab.edu

² Abbreviations used in this paper: TREM, triggering receptor expressed on myeloid cell; EST, expressed sequence tag; FO, follicular; mTLT, mouse TREM-like transcript; MZ, marginal zone; SEB, staphylococcal enterotoxin B; SHP, Src homology region 2 domain-containing phosphatase; T1, transitional 1; T2, transitional 2; TLT, TREM-like transcript.

Received for publication December 29, 2005. Accepted for publication March 1, 2006.

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