Published online before print March 21, 2006
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T cells involves CD36
Veterinary Molecular Biology, Montana State University, Bozeman
1Correspondence: Veterinary Molecular Biology, Montana State University, 960 Technology Blvd., Bozeman, MT 59718. E-mail: uvsmj{at}montana.edu
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T cells express CD36 transcripts. Here, we tested whether bovine T cells express CD36 protein and if so, whether it functions in a manner similar to the monocyte molecule. CD36 transcripts and internal and cell surface protein could be detected in resting, sorted T cells. Phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment increased CD36 transcript levels (detectable at 4 h) and protein expression (internal and cell surface). Increased surface antigen expression was detectable by 24 h and was maximal at 72 h following PMA/ionomycin stimulation. Anti-CD36 monoclonal antibody inhibited increased macrophage-inflammatory protein-1
gene expression in T cells activated by LTA. In conclusion, T cells express CD36, previously thought to be a myeloid and endothelial cell-restricted surface antigen, and it contributes to responses by these cells to microbial LTA.
Key Words: innate • scavenger receptor • Toll-like receptors • PAMP
T cells are the first T cells to develop, can be found in sites of entry into the body (epithelial cell-associated tissues, such as gut and pulmonary mucosa), accumulate during inflammation, and are thought to be involved in innate immune responses against a wide spectrum of pathogens [1
2
3
4
]. In epithelial cell-associated tissues and sites of inflammation, cells of the innate immune system, such as myeloid cells, epithelial cells, dendritic cells, and some specialized T cells, including T cells, can encounter invading microbes via recognition of pathogen-associated molecular patterns (PAMPs) [5
6
7
]. In T cells, PAMPs, such as crude lipopolysaccharide (LPS) preparations, induce selective expression of some chemokines, such as macrophage-inflammatory protein-1 (MIP-1) and MIP-1ß [7
]. In global gene expression analyses, we found that bovine T cells express transcripts for a number of different PAMP receptors, including scavenger receptors (such as CD36), Toll-like receptors (TLRs), and CD11b, among others [7
]. The importance of these receptors in PAMP responses by T cells has not been characterized.
The CD36 PAMP receptor is a member of the scavenger receptor family of leukocyte antigens. It is thought to be restricted to monocytes and endothelial cells and serves as a receptor for oxidized lipids, apoptotic cells, thrombospondin-1, and Plasmodium falciparum-parasitized erythrocytes [8
9
10
]. As mentioned above, CD36 has been shown to facilitate monocyte TLR2 responses against lipoteichoic acid (LTA), analogous to the role CD14 plays in facilitating TLR4 recognition of LPS [10
]. It is interesting that CD36 is quite restrictive in its effect on TLR2 function, enhancing TLR2/TLR6 ligand (LTA) but not TLR2/TLR1 ligand {the synthetic triacylated lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (PAM3CSK4) interactions [10
]. In this report, we tested the hypothesis that CD36 serves a similar function on T cells.
In agreement with our earlier study [7
], CD36 transcripts were detected in resting, sorted T cells (Fig. 1
). As also seen in our earlier study [7
], stimulation of T cells, in this case with PMA/ionomycin for 4 h, increased the level of detectable CD36 mRNA (Fig. 1)
. CD36 protein expression in bovine T cells was examined by fluorescence-activated cell sorter (FACS) and immunohistochemical analyses using an anti-human CD36 monoclonal antibody (mAb), which cross-reacts with bovine CD36 [9
]. Anti-CD36 mAb stained a fraction of resting peripheral blood T cells, which was repeated in cells from four different calves (Fig. 2A
). PMA/ionomycin treatment increased surface expression of CD36 on T cells, which was detectable at 24 h. After 72 h, nearly all T cells were CD36-positive following PMA/ionomycin stimulation (Fig. 2B)
. A few non- T cells also expressed CD36, but this fraction was highly variable between samples, and it is unclear at this time whether they were natural killer cells, a small subset of ß T cells, or even B cells (data not shown). Intracellular CD36 was examined by staining acetone-fixed cytospin preparations of sorted, resting, and 72 h PMA/ionomycin-stimulated T cells (72 h was chosen based on the FACS analyses shown in Fig. 2
). In contrast to the expression of CD36 on only a fraction of resting T cells detected by FACS (Fig. 2)
, most resting T cells in cytospin preparations were CD36-positive, suggesting they expressed CD36 within intracellular granules (Fig. 3
). Following PMA/ionomycin activation for 72 h, there was an obvious increase in staining, suggesting new protein synthesis and based on the FACS analysis (Fig. 2)
, translocation of the intracellular pools to the cell surface (Fig. 3)
. Western blot analysis showed the anti-CD36-reactive molecule on T cells to be the predicted molecular mass of CD36 (
80 kDa, data not shown). These results document that T cells express intracellular and cell surface forms of the CD36 protein and that activation leads to an increase of the cell surface molecule.
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Figure 1. Bovine T cells expressed CD36 mRNA, which increased following phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. T cells were purified from bovine (1–6 month-old males; 17 total for the entire study) peripheral blood mononuclear cell (PBMC) preparations to >95% using an antibody/magnetic cell sorter (MACS) magnetic bead cell separation protocol and cultured as described previously [7
]. The cells were then treated with 20 ng/ml PMA and 0.5 µg/ml ionomycin or phosphate-buffered saline (PBS; resting) for 4 h. RNA was extracted, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed, as described [7
], using bovine-specific CD36 primers. Results reflect means ± SD of three replicates. Results are representative of three individual experiments.
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Figure 2. CD36 surface expression increases on T cells following stimulation with PMA/ionomycin. Two-color flow cytometry was performed using standard protocols, as described previously [4
]. Briefly, cells were stained sequentially with 20 µg/ml anti-CD36 mAb, phycoerythrin (PE)-conjugated goat anti-mouse, 10% mouse serum, and fluorescein isothiocyanate-conjugated GD3.8 (anti-pan T cell). All FACS was done on a BD FACSCalibur using Cell Quest software. (A) CD36 expression on resting T cells from four different animals. (B) CD36 expression on T cells at time zero (B, Panel A) and 12 h (B, Panel B), 24 h (B, Panel C), 48 h (B, Panel D), and 72 h (B, Panel E) after PMA/ionomycin treatment in complete RPMI (10% fetal bovine serum, antibiotics, HEPES). (B, Panels F and G) PE second stage-only and isotype-matched primary mAb, plus PE second stage-negative controls, respectively. Quadrant markers were set based on background staining with second-stage reagent alone. Values reflect the percent of T cells in the upper right quadrant. (B) Results are representative of three individual experiments.
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Figure 3. T cells expressed intracellular and cell surface forms of the CD36 protein. T cells were purified by panning on E-selectin cDNA L-cell transfectants, as described previously [11
], which yielded a preparation of >85% pure T cells without surface antibody, as occurred with the MACS separation protocol described in Figure 1
. Cytospin slide preparations of resting or 72 h PMA/ionomycin-stimulated, purified T cells were prepared, blocked in PBS containing 5% goat serum, and stained with 20 µg/ml anti-CD36 or GD3.8 anti- T cell mAb, and primary mAb was detected by addition of goat anti-mouse peroxidase-conjugated secondary antibody and 3-amino-9-ethylcarbazole developing solution (TAGO, Inc., Fort Wayne, IN). Anti-CD36-stained resting T cells (A), anti-CD36 (B), and GD3.8-stained T cells (C) treated with PMA/ionomycin for 72 h and second stage-alone, negative control (D) are shown. Images were taken at 40x original magnification.
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T cells increase transcript levels for MIP-1 following 4 h stimulation with PAMPs, such as peptidoglycan and LPS [7
] and LTA (unpublished observations). To test whether CD36 contributes to the recognition of LTA by T cells, as recently shown for monocytes [10
], we used the conditions of our earlier assays and tested the effect of anti-CD36 mAb versus an isotype-matched, negative control mAb. As seen before for other PAMPs [7
], T cells from different animals displayed variable responses to LTA, but in each case where LTA induced MIP-1 up-regulation, anti-CD36 mAb, but not the negative control mAb, blocked the effect (Fig. 4
and data not shown). The anti-CD36 mAb effect was specific to TLR2/TLR6 function in that responses to PAM3CSK4 (TLR2/TLR1 agonist) were not affected (Fig. 4)
.
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Figure 4. Anti-CD36 mAb blocked LTA-induced increase in MIP-1 mRNA expression in T cells, which were purified from bovine PBMC preparations to >95% using an antibody/MACS magnetic bead cell separation protocol as described in Figure 1
. Purified T cells were stimulated with 10 µg/ml LTA or 10 µg/ml PAM3CSK4 in the presence or absence of anti-CD36 mAb or an isotype-matched, negative control mAb (IgM, Immunoglobulin M) (10 ug/ml) for 4 h. RNA was extracted, and real-time RT-PCR was performed as described [7
] using MIP-1-specific primers. Results reflect mean ± SD from three replicates and are representative of three different experiments.
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T cells express CD36 at the RNA, protein, and functional level. CD36 is expressed on resting T cells, but surface levels increase considerably following PMA/ionomycin treatment. Consistent with recent findings for monocytes, CD36 facilitates T cell responses to LTA [10
]. This interaction likely requires TLR2/TLR6, although this awaits formal demonstration. Additional studies are under way to test whether T cell CD36 also functions as a receptor for oxidized low-density lipoproteins and/or apoptotic cells, other activities attributed to the monocyte molecule [8
]. Expression of CD36 on T cells is consistent with and provides additional support to the role of these cells in innate immunity.
Received October 28, 2005; revised December 21, 2005; accepted January 12, 2006.
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Cells: a right time and a right place for a conserved third way of protection Annu. Rev. Immunol. 18,975-1026[CrossRef][Medline] T cell subset, which is found in large numbers in the spleen, accumulates inefficiently in an artificial site of inflammation: correlation with lack of expression of E-selectin ligands and L-selectin J. Immunol. 162,4914-4919 T cells respond directly to pathogen-associated molecular patterns J. Immunol. 174,6045-6053/ T cells J. Immunol. 153,3917-3928[Abstract]This article has been cited by other articles:
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