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The Journal of Immunology, 1998, 161: 6265-6272.
Copyright © 1998 by The American Association of Immunologists

Mast Cells Express Novel CD8 Molecules That Selectively Modulate Mediator Secretion1

Tong-Jun Lin2, Nadir Hirji, Osamu Nohara, Grant R. Stenton, Mark Gilchrist and A. Dean Befus

Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD8, a marker largely restricted to subsets of T lymphocytes and NK cells, was detected on freshly isolated rat peritoneal mast cells (PMC). Using flow cytometry, Percoll-enriched rat PMC (>=98% purity) were positive for the hinge region of CD8{alpha} (67.5 ± 9.5%; Ab OX8) and CD8ß (27.8 ± 2.3%; Ab 341). CD8+ PMC consisted of two populations, CD8{alpha}+ (22.5%) and CD8{alpha}+ß+ (15.9%). Interestingly, G28, an Ab that identifies the IgV-like region of CD8{alpha} on T lymphocytes, did not bind PMC, suggesting that PMC CD8{alpha} is distinct from that on T lymphocytes. Moreover, a similar pattern of Ab positivity for CD8 was observed on a rat mast cell line, RBL 2H3. The presence of CD8{alpha} immunoreactivity on rat PMC was further confirmed by confocal microscopy. In situ reverse-transcription PCR and reverse-transcription PCR analysis demonstrated that PMC contained mRNA transcripts encoding CD8{alpha}. In functional studies of CD8 on PMC, both TNF-{alpha} and nitric oxide production were induced by OX8 (CD8{alpha}) and 341 Ab (CD8ß) in a dose-dependent manner. However, neither OX8 nor 341 induced histamine secretion from PMC. Ag-induced secretion of TNF-{alpha}, nitric oxide, and histamine was not affected by OX8 or 341 Abs, suggesting that there are distinct signaling mechanisms mediated by CD8 and Fc{epsilon}RI. These results indicate that rat PMC express functional CD8 molecules that may be distinct from those of T lymphocytes. The difference suggests there is a ligand other than MHC class I for mast cell CD8.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD8 is a cell surface glycoprotein thought to be largely restricted to subsets of suppress/CTL and NK cells. It is expressed as a disulfide-linked {alpha}/{alpha} homodimer or {alpha}/ß heterodimer (in rat {alpha}, 32 kDa; ß, 37 kDa) (1, 2, 3). Both the {alpha}- and ß-chains of CD8 consist of an NH2-terminal domain homologous to IgV domains, a short hinge region connecting the Ig-like domain to a hydrophobic transmembrane segment, and a cytoplasmic tail (1). The cytoplasmic region of the {alpha}-chain includes a binding site for the src-related tyrosine kinase p56lck, through which the cosignaling effects of CD8 are mediated (4). CD8 in cytotoxic T cells functions as an adhesion protein and a cosignaling receptor (5, 6, 7). Recently, we described a novel form of CD8{alpha} and CD8ß expressed on rat macrophages (7). Furthermore, ligation by Abs of CD8{alpha} and CD8ß on macrophages induced both secretion of inflammatory mediators and cytotoxic activity (7). Given our observations and the recent report that a mouse mast cell (MC)3 line expresses CD8{alpha} (8), we investigated the hypothesis that freshly isolated MC express CD8{alpha} and CD8ß and that ligation of these CD8 molecules by Abs would induce MC activation.

MC play important roles in allergic and other inflammatory reactions by producing a spectrum of powerful mediators including preformed (such as histamine), de novo synthesized (such as nitric oxide (NO)), and a wide variety of cytokines (including TNF-{alpha}) (9, 10, 11). Recently, a number of studies have demonstrated that MC-derived TNF-{alpha} represents a central component of host defense against bacterial infection and is crucial for the recruitment of neutrophils in bacterial and immune complex-induced inflammation (12, 13, 14). MC-derived NO is involved in the regulation of MC function in an autocrine manner (15) and has been shown to be important in the maintenance of the gut epithelial barrier (16). Interestingly, MC possess mechanisms to regulate the secretion of mediators differentially (17, 18, 19, 20).

In the present study, we demonstrated for the first time that rat peritoneal MC (PMC) express surface CD8 proteins. The functional roles of these CD8 molecules on mediator secretion from MC were studied. Moreover, no cooperative effects between CD8 and Fc{epsilon}RI on MC activation could be observed. Thus, we suggested that CD8-induced intracellular signal pathways are distinct from that mediated by Fc{epsilon}RI. The biologic significance of MC CD8 was also discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents

Adult male Sprague Dawley (Crl:CD (SD) BR) rats were obtained from Charles River Canada (Quebec, Canada) (7) and infected with Nippostrongylus brasiliensis, as previously described (21), when MC were used for Ag stimulation.

The Abs, OX8 (anti-CD8{alpha} hinge region, IgG1) (22), OX8-FITC, and W3/25-FITC (anti-CD4, IgG1) (22) and OX41 (IgG 2a) were purchased from Serotec (Toronto, Canada). G28 (anti-CD8{alpha} IgV-like region, IgG2a) (23), G28-FITC, 341 (anti-CD8ß, IgG1) (23), and 341-FITC were purchased from Cedarlane (Hornby, Canada). IgG1, IgG1-FITC, IgG2a, and IgG2a-FITC were purchased from Accurate Chemical and Scientific (New York, NY). Rat rSCF was the kind gift from Amgen (Thousand Oaks, CA) (>95% pure by SDS-PAGE). Heparinase I, o-Phthaldialdehyde (OPT), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulfanilamide, and naphthylethylene diamine dihydrochloride were purchased from Sigma (St. Louis, MO). RPMI 1640 medium and HEPES-buffered Tyrode’s solution (HBTS) were purchased from Life Technologies (Grand Island, NY). Ag used to activate in vivo sensitized MC was prepared according to the method described (21).

The Ag concentration was described as worm equivalents (we)/ml, and the final protein concentration of Ag (5 we/ml) was 7.1 ± 0.8 µg/ml. The Ag, RPMI 1640 medium, and HBTS contained less than 0.01 ng/ml of endotoxin when tested by the E-Toxate kit (Sigma). Endotoxin-free water (Baxter, Toronto, Canada) was used in all experiments.

MC isolation, incubation, and histamine assay

PMC were prepared, as described previously (24), with purity of >=98%. In several experiments (long term), PMC were incubated in RPMI 1640 medium with Abs or rat rSCF for 48 h, then washed and resuspended in HBTS and challenged with Ag (5 we/ml). In other experiments (short term), PMC were incubated in HBTS with Abs for 20 min and then challenged with Ag (5 we/ml) without first removing Abs. Histamine was measured in both supernatant and pellet fractions by fluorometric assay (25) using a CytoFluor 2350 Fluorescence Spectrometer (Millipore). Unless specified otherwise, the spontaneous release of histamine in the absence of stimulant (2.5 ± 0.9%, n = 7) was subtracted to establish histamine release specific to the secretogogues employed.

Flow-cytometric analysis

In 96-well U-bottom plates, cells (5 x 105 cells per test) were preincubated in immunofluorescence (IF) buffer (PBS + 1% BSA + 0.2% sodium azide) + 10% normal mouse serum (for conjugated primary Abs only) for 30 min before 1-h Ab incubation at 4°C. Cells were washed three times (with IF buffer) and resuspended in 400 µl of 1% Formalin (IF buffer), and 10,000 cells were analyzed on a FACScan (Becton Dickinson, Mountain View CA). The results with specific Abs were compared with isotype-matched controls.

Confocal microscopy imaging of CD8

In a 96-well U-bottom plate, cells (5 x 105 cells per test) were preincubated in HBTS + 10% normal mouse serum for 30 min before incubation for 1 h with OX8-FITC at 4°C. Cells were washed three times (HBTS) and resuspended in 4% Formalin (HBTS). Cytospins of OX8-labeled PMC were made by vortexing slides in a Cytospin 2 (Shandon, U.K.) at 600 rpm for 3 min. Antibleaching solution (10 mM n-propyl gallate (Sigma), 8.1 M glycerol, in Tris-buffered saline) was dropped onto slides before coverslip attachment. Cells were examined with a Leica confocal laser-scanning microscope (Heidelberg, Germany). The results with OX8-FITC were compared with isotype-matched controls.

Measurement of TNF-{alpha} and NO2-

TNF-{alpha} in cell-free supernatants was tested as cytotoxicity of WEHI 164.13 using MTT assay, as previously described (19, 26). Mouse rTNF-{alpha} (Genzyme, Cambridge, MA) was used as a standard. NO2- in supernatants was measured by Griess reagent (1% sulfanilamine, 0.1% N-(1-naphthyl)-ethylene-diamine dihydrochloride, 2.5% H3PO4) (27). Results were expressed as µM following incubation of 2 x 105 cell in 200 µl volume. NaNO2 was used as a standard. The plate was read on Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA) at 570 nm for TNF-{alpha} and 540 nm for NO2-.

Reverse-transcription PCR

Total RNA was extracted from PMC yielding 1.61 ± 0.36 µg/106 PMC and treated with heparinase to remove contaminating heparin (28). Splenic CD8 T cells isolated by a rat CD8 immunocolumn (Biotex Laboratories, Edmonton, Canada) and a murine tumor cell line, WEHI 164.13, were used as a positive and negative control, respectively. mRNA was reverse transcribed by SuperScript RNase (Life Technologies) according to the manufacturer’s protocols. PCR was performed with some modifications of the Life Technologies Taq DNA polymerase protocol using a PTC-100 Programmable Thermal Controller (MJ Research, Cambridge, MA) (28). The primers used were: 1) rat ß-actin 5' primer, 5'-GTG GGG CGC CCC AGG CAC CA-3', and 3' primer, 5'-GTC CTT AAT GTC ACG CAC GAT TTC-3'; 2) rat CD8{alpha} primer I (Ig-like region) 5' primer (nucleotide 136, within amino acid 5), 5'-TCA CCA AAG AAA GTG GAG GC-3', and 3' primer (nucleotide 370, within amino acid 123), 5'-CTT GCT CAG GGT GAG GAT GT-3'; and 3) rat CD8{alpha} primer II, 5' primer, 5'-CAG TTA CAG TTG TCA CCA AA-3', and 3' primer, 5'-CAC GAA TTT CTC TGA AGG TC-3'. The PCR products for ß-actin, CD8{alpha} primer I (Ig-like), and primer II were 526, 234, and 630 bp, respectively. Thirty-five cycles were used for CD8{alpha} and 25 cycles for ß-actin (95°C for 45 s, 55°C for 45 s, and 72°C for 2 min). Products were run on a 2% agarose gel and stained with EtBr.

In situ RT-PCR

In situ RT-PCR was modified and performed as previously described (29). Briefly, cells were fixed for 16 h in 10% buffered Formalin, washed twice with diethylpyrocarbonate (DEPC)-treated water, then placed on slides and allowed to dry overnight. The cells were digested in 2 mg/ml (5000 U/ml) pepsin (Boehringer Mannheim, Mannheim, Germany) in 0.01 M HCl, then treated with RNase-free DNase I (Boehringer Mannheim) at 37°C overnight. After that, PMC, but not RBL-2H3, were treated with 333 U/ml Heparinase I (Sigma) for 2 h at room temperature (28). The test specimens were treated with a RT solution containing 25 µg/ml oligo(dT)12–18 primer (Life Technologies/BRL) with MMLV-RT (Life Technologies/BRL) as the enzyme for 2 h at 37°C. Amplification of the cDNA was accomplished with a PCR solution containing 4.5 mM MgCl2; 80 µM each of dATP, dCTP, dGTP, and dTTP; 0.8 µM of each primer (CD8{alpha} primer set I); 16 µM digoxigenin-11-dUTP (Boehringer Mannheim); and 120 U/ml Taq polymerase (Life Technologies/BRL). Cycling conditions were 5 min at 94°C and 30 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1.5 min. The digoxigenin-11-dUTP-labeled PCR product was detected after incubation with alkaline phosphatase antidigoxigenin conjugate (Boehringer Mannheim) (1/200 dilution in 0.1 M Tris-HCl, 0.15 M NaCl, pH 7.5) for 30 min at room temperature, and development in NBT/BCIP (Boehringer Mannheim) substrate solution. Positive controls to monitor the length of pepsin digestion showed nuclear DNA priming, and negative controls to monitor DNase digestion showed that no priming of genomic DNA was detectable.

Statistical analysis

Analysis of variance and the paired Student’s t test were used for statistical evaluation of data. Results are considered significantly different when p < 0.05. Throughout the text, data are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Flow cytometry and confocal microscopy of CD8 expression on rat PMC

To test the possibility that PMC express CD8 molecules, mAbs, OX8, which recognizes CD8{alpha} hinge region; G28, which identifies the CD8{alpha} IgV-like region; and 341, which identifies CD8ß, were used. Surprisingly, the majority of PMC (67.5 ± 9.5%, n = 4) were OX8 (CD8{alpha}) positive (Figs. 1Go and 2a). There were also a significant number of 341 (CD8ß)-positive cells (27.8 ± 2.3%, n = 4). Interestingly, few PMC were positive for G28 (7.3 ± 1.1%, n = 4) as compared with OX8, unlike T lymphocytes, which were similarly positive for both OX8 and G28, 79 ± 1% and 73 ± 3%, respectively. Few PMC were W3/25 (CD4) positive (2 ± 0.9%, n = 3).



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  		FIGURE 1. Detection of CD8{alpha} and CD8ß on rat PMC by flow cytometry analysis. Freshly isolated rat PMC were stained with OX8-FITC (CD8{alpha}) (a), 341-FITC (CD8ß) (b), or isotype control IgG1-FITC.

 
To examine the expression of CD8 on other MC populations, the same Abs (OX8, G28, and 341) were used to determine the presence of CD8 on RBL 2H3 cells, a rat MC line with some properties of mucosal mast cells (30) (Fig. 2Gob). A large proportion of RBL 2H3 cells were OX8 (CD8{alpha}) and 341 (CD8ß) positive, 87.7 ± 1.9% and 81.7 ± 1.9%, respectively (n = 3). By contrast, only a small proportion of RBL 2H3 cells were G28 (CD8{alpha} Ig-like domain) positive (5.7 ± 3%, n = 3), as seen with PMC.



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  		FIGURE 2. The abundance of OX8 (CD8{alpha} hinge region)-, G28 (CD8{alpha} Ig-like domain)-, 341 (CD8ß)-, and W3/25 (CD4)-positive cells in rat PMC (a) and RBL 2H3 (b). Results are mean ± SEM for four (a) and three (b) experiments.

 
To further confirm the presence of CD8 on rat PMC, Percoll-enriched PMC were stained with OX8 (CD8{alpha}) or isotype-matched IgG1 (control) and examined by confocal microscopy. Consistent with the flow-cytometric data, rat PMC were positive for OX8 (CD8{alpha}) (Fig. 3Go).



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  		FIGURE 3. Confocal microscopic analysis of rat PMC by OX8 (CD8{alpha}) staining. Rat PMC were stained with OX8 or isotype-matched control Ab IgG1.

 
As PMC are positive for both CD8{alpha} (OX8) and CD8ß (341), we stained these cells with OX8-PE and 341-FITC Abs to determine whether CD8{alpha} and CD8ß are expressed on the same cells. Staining, compared with isotype controls, showed that CD8-positive PMC were composed of two populations, CD8{alpha}+ (22.5%) and CD8{alpha}+ß+ (15.9%), with virtually no single ß-chain (0.5%)-positive cells (Fig. 4Go). These experiments were done approximately 8 mo after others described in this work. We observed a lower number of CD8ß-positive cells in the later experiments, an observation for which we have no clear understanding.



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  		FIGURE 4. Double staining of PMC for CD8{alpha} (OX8-PE) and CD8ß (341-FITC), compared with isotype controls (IgG1-PE and IgG1-FITC).

 
In situ RT-PCR and RT-PCR analysis of CD8 expression by rat MC

Given the capabilities of phagocytosis (31) and Ag presentation (32) of MC, one possible explanation for the presence of CD8 on PMC could be that these cells phagocytose exogenous CD8 and reexpress it on their surface. Alternatively, rat PMC may synthesize CD8 themselves. To examine this possibility, in situ RT-PCR analysis was conducted. Although the intensity of the positive signal in the cytoplasm varied among PMC, 94.7% of PMC (two independent experiments; 500 cells counted/experiment) were positive for CD8{alpha} Ig-like domain (Fig. 5Goa). The RBL-2H3 cells showed a strong positive signal in all cells (Fig. 5God). Interestingly, CD8{alpha} mRNA was localized in the perinuclear region of PMC, whereas CD8{alpha} mRNA in RBL-2H3 was detected throughout the cytoplasm, albeit with a strong positive signal near the nucleus in some cells. The nuclei were unstained, reflecting the removal of genomic DNA by DNase treatment. The positive control with nuclear staining demonstrates that pepsin treatment was optimal and subsequent PCR steps were successful (Fig. 5Go, b and e). In the negative control, in which the cells were treated with DNase and the RT step was eliminated, the cells were unstained, indicating that the product detected was indeed cDNA and not priming from contaminating genomic DNA (Fig. 5Go, c and f).



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  		FIGURE 5. Localization of CD8{alpha} Ig-like domain mRNA in Formalin-fixed smears of PMC (a, b, and c) and RBL-2H3 cells (d, e, and f) by in situ RT-PCR. As shown in a and d, several cells displayed distinct cytoplasmic staining (arrow) with greater intensity in perinuclear region (arrowhead). The nuclei are unstained, reflecting the removal of genomic DNA by DNase treatment. In b and e, all cells showed intense nuclear staining (arrow) in the positive control, indicating nuclear DNA priming and that pepsin treatment was optimal, thereby allowing PCR reagents to enter cells. In c and f, in which the cells were treated with DNase and the RT step was eliminated, no staining was shown in the negative control, indicating that the product detected was indeed cDNA and not priming from contaminating genomic DNA. Original magnification: x1000 (a, b, c); x400 (d, e, f), bar = 10 µm.

 
To further confirm the expression of CD8 by MC, we used RT-PCR to detect mRNA for CD8{alpha} in PMC. Total RNA from enriched splenic CD8 lymphocytes (81 ± 3% OX52 (T lymphocytes), 79 ± 1% OX8 (CD8{alpha}), 73 ± 3% G28 (CD8{alpha}), and 63 ± 4% 341 (CD8ß)) was used as a positive control. After establishing the presence of a positive RT reaction using paper chromatography, cDNA from Percoll-enriched rat PMC (>=98%) and splenic CD8 lymphocytes were used in the PCR reaction and tested with two sets of PCR primers for CD8{alpha}. One set of primers was designed to detect the Ig-like domain of CD8{alpha} (CD8{alpha} primer set I), which yields 234-bp PCR product. The other (CD8{alpha} primer set II) (33) yields 630-bp PCR product. As shown in Fig. 6Go, rat PMC were positive, using both sets of primers for CD8{alpha}, as were T lymphocytes. cDNA generated from WEHI 164.13 cells or water were employed as a negative control, and no PCR products could be found (data not shown).



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  		FIGURE 6. PCR analysis of rat PMC (>=98% pure) for CD8{alpha} (primer I, 234 bp; primer II, 630 bp), compared with CD8--enriched splenic T lymphocytes.

 
Although the Ig-like domain appears to be lacking or is masked in the CD8{alpha} protein expressed on PMC, the PCR primers, which included this region, detected mRNA in PMC, as we recently described in rat macrophages (7).

TNF-{alpha} production by CD8{alpha} (OX8)- and CD8ß (341)-stimulated rat PMC

To assess the functional significance of CD8 on PMC, we stimulated PMC with OX8 or 341 at the dose of 0.5, 2, or 5 µg/ml for 6 h, and tested TNF-{alpha} production in the cell-free supernatants. OX8 and 341 stimulated TNF-{alpha} production by PMC in a dose-dependent manner (Fig. 7Go). There was a significant increase in TNF-{alpha} production following treatment with OX8 (CD8{alpha}) at 2 and 5 µg/ml when compared with isotype control IgG1 (p < 0.01, n = 11) (Fig. 7Goa). Similarly, mAb 341 (CD8ß) stimulated TNF-{alpha} production significantly at 2 and 5 µg/ml compared with isotype control IgG1 (p < 0.01, n = 7) (Fig. 7Gob). Interestingly, no additive or synergistic effects between OX8 and 341 could be found at any of the doses tested (0.5, 2, or 5 µg/ml). TNF-{alpha} production by rat PMC was not affected by G28 (data not shown), as expected, given the absence of the respective epitope on CD8{alpha} on mast cells (see above).



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  		FIGURE 7. OX8 (CD8{alpha}) and 341 (CD8ß) stimulate TNF-{alpha} production by rat PMC. The cells were incubated with OX8 (a), 341 (b), or IgG1 for 6 h. TNF-{alpha} levels in cell-free supernatants were determined. Results are mean ± SEM for 11 (a) or 7 (b) experiments. (*, p < 0.05 by comparison with IgG1 or sham-treated cells.)

 
NO production by CD8{alpha} (OX8)- and CD8ß (341)-stimulated rat PMC

To assess the effects of CD8 on NO production by PMC, we incubated PMC (1 x 106 PMC/ml) with OX8, 341, or isotype control IgG1 at the doses of 0.5, 2, or 5 µg/ml for 48 h. Nitrite (as an indicator of NO) production by PMC in the cell-free supernatants was tested. As shown in Fig. 8Go, OX8 and 341 each significantly stimulated nitrite production. There was a significant increase (p < 0.05, n = 9) in nitrite production with 2 and 5 µg/ml OX8 compared with isotype control IgG1 (Fig. 8Goa). Similarly, 341 (CD8ß) significantly stimulated NO production at the dose of 5 µg/ml (6.9 ± 1.4 µM nitrite) compared with IgG1 (5 ± 0.7 µM nitrite) (p < 0.05, n = 6) (Fig. 8Gob).



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  		FIGURE 8. OX8 (CD8{alpha}) and 341 (CD8ß) stimulate NO production by rat PMC. The cells were incubated with OX8 (a), 341 (b), or IgG1 for 48 h. Nitrite in cell-free supernatants was determined. Results are mean ± SEM for 11 (a) or 6 (b) experiments. (*, p < 0.05 by comparison with IgG1 or sham-treated cells.)

 
To exclude the possibility that the binding of any Ab to MC surface might influence TNF-{alpha} or NO production, OX41 Abs (93% PMC were OX41 positive, n = 5) were used to treat PMC for 6 h (TNF-{alpha}) or 48 h (NO) at the dose of 5, 10, and 50 µg/ml. Also, the potency of OX8 and 341 in stimulation of TNF-{alpha} or NO production was compared with that of sensitizing Ag. Our results demonstrated that OX41 did not stimulate TNF-{alpha} or NO production (p > 0.05, compared with no treatment group, n = 4–8). Interestingly, TNF-{alpha} and NO production mediated by OX8 and 341 were comparable with that induced by optimal dose of Ag (5 we/ml, Table IGo).


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  		Table I. OX8 (CD8{alpha}) and 341 (CD8ß) and Ag but not OX41 stimulate TNF-{alpha} and NO production by rat peritoneal mast cells

 
No effects of CD8{alpha} (OX8) and CD8ß (341) on histamine secretion by rat PMC

We tested the effects of CD8 on the secretion of the preformed mediator, histamine, from PMC. Cells were incubated with OX8 (CD8{alpha}) or 341 (CD8ß) at doses of 2, 5, or 10 µg/ml for 30 min. Histamine secreted in cell-free supernatants as well as stored in the cell pellets was determined. As shown in Fig. 9Go, there was no direct stimulation of histamine secretion by OX8 (CD8{alpha}) or 341 (CD8ß) at 2, 5, or 10 µg/ml compared with isotype control IgG1 (p > 0.05, n = 3) (spontaneous histamine release: 1.7 ± 0.6%).



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  		FIGURE 9. No effects of OX8 (CD8{alpha}) and 341 (CD8ß) on histamine secretion from rat PMC. The cells were incubated with OX8, 341, isotype control IgG1, or Ag (5 we/ml) for 30 min. Histamine secretion was determined. Results are mean ± SEM for three experiments. (*, p < 0.05 by comparison with sham-treated cells.)

 
No effects of CD8 on Ag-induced mediator secretion from PMC

To investigate possible interactions between signaling from CD8 and Fc{epsilon}RI, effects of ligation of CD8 on Ag-induced mediator secretion from PMC were studied. To determine effects of CD8 on TNF-{alpha} and NO production, PMC were incubated with Ag (5 we/ml) together with OX8, 341, or isotype control IgG1 at the doses of 0.5, 2, or 5 µg/ml for 6 or 48 h, respectively; then cell-free supernatants were used for TNF-{alpha} and NO assays. Although both CD8{alpha} and CD8ß directly stimulated TNF-{alpha} and NO production (Figs. 7Go and 8Go), Ag-induced TNF-{alpha} and NO production by PMC was not affected by OX8 at the dose of 0.5, 2, or 5 µg/ml (p > 0.05, n = 4) (Fig. 9Go, a and b). Similarly, 341 (CD8ß) at the dose of 0.5, 2, or 5 µg/ml did not affect Ag-induced TNF-{alpha} and NO production (p > 0.05, data not shown).

To examine effects of CD8 on Ag-induced histamine secretion, PMC were incubated with OX8 (CD8{alpha}), 341 (CD8ß), or isotype control IgG1 at the dose of 0.5, 2, or 5 µg/ml for 48 h. After washing, rat PMC were stimulated with Ag (10 we/ml) for 10 min. No effects of OX8 (CD8{alpha}) or 341 (CD8ß) were observed on Ag-induced histamine secretion from PMC (p > 0.05, n = 4) (Fig. 10Goc). Spontaneous histamine secretion from PMC was not affected by incubation for 48 h with OX8 (CD8{alpha}), 0.5 (2.7 ± 0.3%), 2 (2.3 ± 0.9%), and 5 (2.6 ± 0.6%) µg/ml, or 341 (CD8ß), 0.5 (2.9 ± 0.9%), 2 (3.3 ± 1.9%), and 5 (2.6 ± 1.6%) µg/ml when compared with isotype control IgG1, 0.5 (2.8 ± 1.3%), 2 (2.2 ± 0.2%), or 5 (2.7 ± 1.1%) µg/ml (p > 0.05, n = 4). Spontaneous and Ag-induced histamine secretion was not affected by G28 (5 µg/ml) (spontaneous release, no G28, 2.8 ± 0.9%, and with G28, 2.2 ± 0.8%; Ag-induced release, no G28, 22.4 ± 5.1%, and with G28, 24.2 ± 6.1%, p > 0.05, n = 3). As a positive control, SCF (100 ng/ml) significantly potentiated Ag-induced histamine secretion (Ag alone, 22.4 ± 5.1%; Ag + SCF, 57.2 ± 6.5%, p < 0.05, n = 3).



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  		FIGURE 10. No effects of OX8 (CD8{alpha}) and 341 (CD8ß) on Ag-induced mediator secretion from rat PMC. The cells were incubated with OX8, 341, or isotype control IgG1 for 6 or 48 h; cell-free supernatants were used for TNF-{alpha} (6-h) or NO (48-h) assays. For histamine assay, after 48-h incubation with Abs, PMC were washed and challenged with Ag (10 we/ml) for 10 min. Histamine secretion was determined. Results are mean ± SEM for five experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
For the first time, we identified that both freshly isolated rat PMC, a connective tissue type MC, and a rat MC line with mucosal type properties, RBL 2H3, express CD8 mRNA and proteins. In situ RT-PCR analysis provided compelling evidence that CD8 message is expressed by both rat PMC and RBL 2H3 cells (Fig. 5Go). Flow cytometry of purified (>=98%) PMC and RBL 2H3 MC line identified a large number of positive cells for the hinge region of CD8{alpha} (OX8) (PMC, 68%; RBL 2H3, 88%), but a significantly lower percentage (PMC, 7%; RBL 2H3, 6%) of cells positive for the Ig-like region of CD8{alpha} (G28). Interestingly, this differed from CD8-enriched splenic T cells that showed similar positivity for both OX8 (CD8{alpha} hinge, 79%) and G28 (CD8{alpha} Ig-like region, 73%). This disparate pattern of OX8 versus G28 positivity was similar to that seen in alveolar and peritoneal macrophages, as described recently (7). These data together with RT-PCR analysis data, in which the primers used for the detection of CD8{alpha} mRNA encompassed the Ig-like domain, led us to postulate that Ig-like region in the CD8{alpha} protein on rat PMC and RBL 2H3 MC line differs from that on T cells, but is similar to that on macrophages.

It is well established that human and rodent MC are heterogeneous in morphology and function (11, 34, 35). Interestingly, OX8 (CD8{alpha})-positive PMC (68%) did not make up the 98% PMC in our cell populations, indicating that there are CD8+ and CD8- mast cells. Clarification of the differential expression of CD8 molecules in different MC populations and determination of possible functional differences between CD8+ and CD8- MC, as that seen in NK cells (36, 37), may help to further understand MC heterogeneity.

CD8 molecules are expressed as either CD8{alpha}/ß heterodimers or CD8{alpha}/{alpha} homodimers in T lymphocytes (1). These two forms of CD8 dimers are functionally different (38). In PMC, a connective tissue type mast cell, flow cytometry showed that the percentage of OX8 (CD8{alpha})-positive cells was significantly greater than that of 341 (CD8ß)-positive cells (p < 0.05, n = 4) (Fig. 1Goa), suggesting there may be both CD8{alpha}/{alpha} and CD8{alpha}/ß on PMC. This was confirmed when cells were double stained for CD8{alpha} and CD8ß, which showed that PMC can be subdivided into three groups, CD8{alpha}-ß-, CD8{alpha}+, or CD8{alpha}+ß+ (Fig. 4Go). Anti-CD8ß stimulates equivalent levels of TNF-{alpha} and NO secretion compared with anti-CD8{alpha}. One reason may be that the ß signal is transmitted in a CD8{alpha}-dependent manner. In addition, CD8-mediated effects may be {alpha}+ß+ dependent, suggesting that both chains are required for CD8 function. It will be essential to sort cells according to their CD8 expression (homo- or heterodimer) to determine whether they differ functionally. Interestingly, in RBL 2H3, a cell with some properties of mucosal MC, the proportion of OX8 (CD8{alpha})-positive cells was similar to that of 341 (CD8ß)-positive cells (Fig. 2Gob), indicating that it is more likely that there are CD8{alpha}/ß heterodimers on these cells.

Recent studies demonstrated that MC-derived TNF-{alpha} is essential in host defense against bacterial infection (12, 13). Regulation of mediator secretion from MC is mediated by microenvironmental factors through cell surface receptors, such as Fc{epsilon}RI and SCF receptor, c-kit. Our functional studies demonstrated that CD8 on PMC selectively modulates mediator secretion. Ligation of CD8{alpha} or ß directly stimulated TNF-{alpha} and NO production, but did not affect histamine secretion. Interestingly, the selective modulation of different mediator secretion from MC is also observed by other MC surface moieties, such as c-kit (19) or IFN receptors (20).

In T cells, the cosignaling roles of CD8 are mediated by the CD8{alpha} cytoplasmic domain-associated p56lck, which phosphorylates the {zeta} of the TCR complex (5). Although the {gamma}-chain of the abundant high affinity FcR for IgE (Fc{epsilon}RI) in MC is highly homologous to the {zeta}-chain of the TCR (39), no cosignaling effects between CD8 and Fc{epsilon}RI were observed in rat PMC. Our results showed that Ag-induced histamine secretion and NO and TNF-{alpha} production were not modified by OX8 (CD8{alpha}) or 341 (CD8ß) although constitutive NO and TNF-{alpha} production was significantly stimulated by OX8 and 341. Thus, it appears that the intracellular signal-transduction pathways mediated by CD8 are distinct from that by Fc{epsilon}RI. However, given the significant roles of p56lck in CD8-mediated effects in T cells and its physical and functional association with Fc{gamma}RIII (40), the possibility of interactions of CD8 with other FcR on MC cannot be ruled out.

CD8 acts as coreceptor and adhesion molecule in T cell activation. The Ig-like domain of CD8 binds to the nonpolymorphic regions of class I MHC molecule, whereas the TCR recognizes peptide Ags in conjunction with the polymorphic region of MHC molecules (41, 42, 43). Interestingly, if the Ig-like domain is missing, or masked by glycosylation or in another manner on most of the CD8{alpha} molecules expressed by rat PMC, as we have also postulated for rat macrophages, there could be a previously unknown ligand for CD8{alpha} on rat PMC, or alternative sites on CD8{alpha} for interaction with MHC I. It has been postulated by Li et al. (44) that there is a new CD8 ligand (non-class I molecule) expressed on the epithelial cell surface that activates CD8 lymphocytes. Although no ligand for CD8 other than MHC I has been found, ligands other than MHC II have been reported for the other T cell coreceptor, CD4. McCarthy et al. suggested that a ligand for CD4 distinct from MHC II is present in the thymus and is responsible for interacting with CD4 to send the signal necessary for TCR {zeta}-chain phosphorylation (45). Moreover, several laboratories have reported that HIV gp120 serves as a ligand for CD4 or for the CD4 molecule with its sequence in the C-terminal replaced by the CD8 hinge, transmembrane, and cytoplasmic domain (1, 46). Recently, IL-16 has been suggested to be a CD4 ligand also (47). Given similarities in the structures and functions of CD4 and CD8, it would not be surprising if a ligand for CD8, distinct from MHC I, was found.

Expression of CD8{alpha} by MC was also observed by Hara et al. in a murine MC line (8). However, our results differ from Hara et al. in that CD8ß was not expressed by the murine MC/9 variant they studied. This discrepancy could be due to some cell culture artifact, or to the species difference. Thus, further characterization of CD8 molecules on freshly isolated MC from human, mouse, and other species in addition to rats will be important to understand the significance of our observation. It has been reported recently that human B lymphocytes are able to express CD8 (48), and thus, together with our recent finding that macrophages express CD8 (7), it is clear that a broad spectrum of cells other than T cells and NK cells is able to express CD8. Further studies of the distribution and function of CD8 will provide new insights into the biologic roles of this important molecule.

In summary, we have determined that freshly isolated rat PMC express functional CD8 molecules on their surface. Flow cytometry and confocal microscopy have identified that many rat PMC and RBL 2H3 cells are OX8 (CD8{alpha}) and 341 (CD8ß) positive. Moreover, RT-PCR analysis confirmed the presence of CD8{alpha} mRNA in rat PMC. Functional studies demonstrated that CD8 on rat PMC selectively regulates mediator secretion. These results demonstrate significant roles of CD8 on MC.


   Acknowledgments
 
We thank Dr. Bruce Ritchie for the assistance in polymerase chain reaction analysis. Mark Gilchrist, Rene Dery, and Jennifer Pick helped in animal infection. Dr. Fiona L. Wills provided the RBL 2H3 cells. We also thank Dr. Vera Chlumechy for her assistance with confocal microscopy, and Drs. Elyse Bissonnette and Redwan Moqbel for their ongoing advice, helpful comments, and review of the manuscript.


   Footnotes
 
1 This work was supported by Medical Research Council of Canada and Alberta Lung Association grants to A.D.B., who holds the Astra Chair in Asthma Research (University of Alberta), and conducted in the Glaxo-Heritage Asthma Research Laboratory. Back

2 Address correspondence and reprint requests to: Dr. Tong-Jun Lin, Department of Pathology, Dalhousie University, Suite 7G, Sir Charles Tupper Building, Halifax, Nova Scotia, Canada, B3M 4HF. Back

3 Abbreviations used in this paper: MC, mast cell; HBTS, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Tyrode’s solution; IF, immunofluorescence; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; PE, phycoerythrin; PMC, peritoneal mast cell; RT, reverse-transcription; SCF, stem cell factor; we, worm equivalent. Back

Received for publication April 7, 1997. Accepted for publication July 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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