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Activation of Macrophage CD8: Pharmacological Studies of TNF and IL-1ß Production¹

Tong-Jun Lin^2,*, Nadir Hirji^2,, Grant R. Stenton^2,, Mark Gilchrist, Brock J. Grill, Alan D. Schreiber and A. Dean Befus^3,

^* Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada; Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; and University of Pennsylvania School of Medicine, Philadelphia, PA, 19104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1ß and promoted TNF and IL-1ß secretion. Similarly, OX8 Ab (CD8) stimulated NR8383 cells to secrete TNF, IL-1ß, and NO. Activation of CD8ß (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1ß and stimulated secretion of TNF, but not IL-1ß. Interestingly, anti-CD8 Abs did not stimulate IFN- or PGE₂ production, or phagocytosis by macrophages. OX8 (CD8)-induced TNF and IL-1ß production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1ß, stimulation by CD8 is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1ß production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 (OX8)-induced TNF and IL-1ß production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1ß expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alveolar macrophages are the most abundant cell type in the airways and are thought to be important in host defense through the release of various cytokines such as TNF- and IL-1ß. TNF- and IL-1 are often implicated as key mediators in the biological response to bacterial LPS, infection, and inflammatory stimuli (1). TNF- and IL-1 share many biologic activities, in particular that of endogenous pyrogen and an ability to stimulate T cell proliferation (2, 3) and resistance to a number of pathogens including Pneumocystis carinii, etc. (4, 5, 6). Excessive production of TNF- and IL-1ß, primarily by macrophages, is thought to be responsible for LPS-induced lethal toxicity (7, 8). Indeed, many of the toxic effects of LPS, such as adult respiratory distress syndrome and vascular leak syndrome, can be mimicked by TNF- and IL-1ß (9, 10). Production of TNF and IL-1ß by macrophages can be regulated in a similar manner. For example, both can be stimulated by LPS and inhibited by IL-10 (11). Or both can be distinctly modulated; for example, TGF-ß inhibited TNF, but not IL-1ß production (11). Protein tyrosine phosphorylation and protein kinase C (PKC)⁵ are necessary for the production of TNF and IL-1ß (12, 13).

CD8 is a cell surface glycoprotein composed of two disulfide-linked chains that form either a homodimer () or heterodimer (ß). In T cells, the cytoplasmic region of the -chain includes a binding site for the Src-related tyrosine kinase p56^lck, through which the cosignaling effects of CD8 are mediated (14). Once activated, CD8-associated p56^lck activates phospholipase C (PLC) by direct tyrosine phosphorylation to generate a costimulatory signal leading to phosphatidylinositol (PI) hydrolysis and subsequent PKC activation and calcium mobilization (14).

Recently, we observed that rat alveolar and peritoneal macrophages express a novel CD8 molecule that stimulates NO production (15). However, the mRNA expression for CD8 has not been directly associated with macrophages, and the effects of CD8 on cytokine production and their mechanisms have not been studied. In this study, using RT-PCR and flow-cytometry analysis, we demonstrated the expression of CD8 mRNA and protein on an alveolar macrophage cell line (NR8383). Moreover, we focused on TNF and IL-1ß, two major macrophage-derived cytokines, and identified that CD8 activation stimulated TNF and IL-1ß production. The signaling mechanisms for CD8 stimulation were examined using inhibitors to protein tyrosine kinase (PTK; genistein), Src-family tyrosine kinases (PP1), PKC (Ro 31-8220), and PI-3 kinase (wortmannin). In addition, we investigated whether Syk kinase is involved in CD8-dependent mediator release. In T lymphocytes, ZAP 70, a homologue of Syk, is involved in the CD8-TCR signaling cascade (16). In macrophages, recent studies have shown that Syk kinase plays distinct roles in macrophage signaling for NO, TNF, and IL-1ß production and phagocytosis (17, 18, 19). Therefore, we used antisense to Syk to determine whether this kinase is involved in CD8-mediated signaling in macrophages.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Adult male Sprague Dawley rats (200 to 300 g) were obtained from Charles River Canada (Quebec, Canada) and maintained in filter-topped cages to minimize unwanted infections. The animals were given food and water ad libitum and maintained on a 12-h (0700 h)/12-h (1900) light/dark cycle. To minimize the effects of stress associated with transport, new housing facilities, and handling, animals were used only after 1 wk of arrival. All experimental protocols were approved by the Health Sciences Laboratory Animal Services, University of Alberta in accordance with the guidelines of the Canadian Council on Animal Care.

Reagents

The Abs OX8 (anti-CD8 hinge region, IgG1) (20), OX8-FITC, W3/25-FITC (anti-CD4, IgG1) (20), and OX-41-FITC (recognizing an incompletely characterized, 110- to 120-kDa cell surface protein, IgG2a) were purchased from Serotec (Toronto, Canada). G28 (anti-CD8 Ig variable-like region, IgG2a) (21), 341 (anti-CD8ß, IgG1) (20), and 341-FITC were purchased from Cedarlane (Hornby, Canada). IgG1, IgG1-FITC, and IgG2a were purchased from Accurate Chemical and Scientific (New York, NY). LPS, MTT, wortmannin, sulfanilamide, and naphthylethylene diamine dihydrochloride were purchased from Sigma (St. Louis, MO). Genistein and PP1 were purchased from Calbiochem-Novabiochem (San Diego, CA). Ro 31-8220 was a generous gift from Roche Products (Welwyn Garden City, U.K.) and dissolved in 10% DMSO at 1 mM. RPMI 1640 medium was purchased from Life Technologies (Grand Island, NY).

Isolation of bronchoalveolar macrophages

Animals were anesthetized by i.p. injection of 0.5 ml of Rompun (xylazine) and 0.5 ml Ketalean (ketamine). The trachea was catheterized with a polyethylene tube, and 5 x 12 ml of cold PBS was massaged into the lungs. Lavage cells were pelleted at 200 x g for 20 min and resuspended in PBS (22). To further purify alveolar macrophages, alveolar lavage cells were incubated with OX41-FITC and sorted on a Coulter EPICS Elite cell sorter (Coulter Electronics, Hialeah, FL). The enriched population contained <1% OX52 (T lymphocytes) and 89 ± 1% OX41 (15). OX41-FITC did not induce TNF or NO production by alveolar macrophages (data not shown). The percentage of alveolar macrophages, tested by esterase staining, was 96 ± 0.4%.

TNF bioassay

The bioactivity of TNF was tested as cytotoxicity of WEHI 164.13 using MTT assay as previously described (23). Briefly, 50 µl/well of standards or samples was added into flat-bottom Linbro plates. Each sample was tested in four, 2-fold serial dilutions. Mouse recombinant TNF (Genzyme, Cambridge, MA) was used as a standard. Eight 2-fold serial dilutions starting from 100 pg/ml were used to establish the standard curve. Fifty microliters per well of 1x10⁵ WEHI 164.13 cells/ml in RPMI 1640 medium, supplemented with 10% FBS and 50 µM 2-ME, was added and incubated for 20 h. Then, 10 µl/well of MTT (5 mg/ml) was added and further incubated for 3 h. Isopropanol-HCl (150 µl) was used to dissolve the purple formazan precipitates. The plate was read on Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA) at 570 nm. The specific cytotoxicity mediated by TNF was confirmed by using polyclonal rabbit anti-mouse TNF neutralizing Ab (Genzyme). Abs (OX8, 341, or IgG1) and kinase inhibitors (PP1, genistein, wortmannin, or Ro 31-8220) did not interfere with the TNF assay at the doses used. DMSO (0.1%) did not induce TNF production by alveolar macrophages.

To test the specificity of TNF-mediated cytotoxicity in the bioassay, anti-TNF neutralizing Ab was used and completely inhibited the target cell cytotoxicity of supernatants from OX8 or 341 treated cells (data not shown).

ELISA assay for IL-1ß, PGE₂, IFN-, and PTK activity

IL-1ß protein (R&D Systems, Minneapolis, MN), PGE₂ (Cayman Chemical, Ann Arbor, MI), and IFN- (BioSource, Camarillo, CA) contents from cell-free supernatants were measured according to the manufacturer’s instruction using ELISA systems.

For measuring PTK activity, NR8383 cells (1 x 10⁶ cells/ml, 100 µl) were pretreated with or without genistein (10 µg/ml) for 30 min. After three washes, cells were stimulated with mouse IgG1 or OX8 (10 µg/ml) for 5 min and lysed by three cycles of freezing and thawing in extraction buffer (20 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 1 µg/ml pepstatin, 0.5 µg/ml leupeptin, 0.2 mM Na₃VO₄, and 5 mM mercaptoethanol). Cell lysates (10 µl) were used for measuring PTK activity according to the manufacturer’s protocol (Calbiochem-Novabiochem) using ELISA system.

Phagocytosis assays

Sheep erythrocytes (ICN Pharmaceuticals, Aurora, OH) were unopsonized, or opsonized (24), using a polyclonal rabbit IgG anti-sheep erythrocyte (ICN Pharmaceuticals). One hundred microliters of sheep erythrocytes (1% suspension) and 100 µl of NR8383 (4 x 10⁶ cells/ml) were mixed (target to effector ratio of 440:1). The pelleted mixture was incubated (37°C, 1 h), and the free erythrocytes were lysed by adding 100 µl of ammonium chloride lysis buffer for 4 to 5 min, followed by neutralization with Ham’s F-12 media (Life Technologies; Ref. 20). Following preparation of cytocentrifuge smears and staining with Leukostat (Fischer Scientific, Nepean, ON), percentage phagocytosis (percentage of NR8383 with ingested erythrocytes) was scored by microscopy, and the phagocytic index (mean number of erythrocytes/NR8383 cell) was determined.

NR8383 were treated concurrently, or for 8 or 20 h with OX8 or IgG1 isotype control at 5 µg/ml. For pretreatment of 8 or 20 h, NR8383 were suspended at 5 x 10⁵ cells/ml, but resuspended at 4 x 10⁶ cells/ml for phagocytosis assay.

Measurement of NO₂^- production

Macrophages (1 x 10⁵ cells/test) were incubated with OX8 or 341 Abs or isotype controls for 24 h at 37°C, 5% CO₂. Cell-free supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamine, 0.1% N-(1-naphthyl)-ethylene-diamine dihydrochloride, 2.5% H₃PO₄) and incubated for 10 min at room temperature (25). Concentration of NO₂^- was determined by measuring the absorbance at 540 nm with a Molecular Devices Vmax Kinetic Microplate Reader. NaNO₂ was used as a standard.

Syk antisense

Rat Syk antisense was designed as described by Ref. 19 and consisted of the following sequence: 5'-GCCGCGGTTGCCCGCCATGTCTGATTTGATTCTTGAGATTTGGTAGTATCCCTCCGCGGC-3'. The scrambled oligonucleotide control contains the same percentage of A,T,G,C nucleotides within a 60-base sequence.

Syk antisense was incorporated into liposomes, which were prepared as described earlier (26). Briefly, 1 mg of DOTAP (1,2,-dioleoyl-3-trimethylammonium-propane) and 1 mg DOPE (dioleoylphosphatidyl-ethanol-amine), which were both solubilized in chloroform, were mixed, and the chloroform was evaporated to leave a 1:1 DOTAP:DOPE mixture. This was resuspended in saline to form liposomes. Syk antisense or scrambled control oligonucleotides were mixed with liposomes, at a 2.5:1 liposome to nucleotide ratio, to yield a nucleotide-liposome complex.

Syk antisense was aerosolized and administered to rats. Briefly, rats were placed in plexiglass chambers, with liposome, with scrambled nucleotide + liposome, or with Syk antisense + liposome, administered by nebulization for 30 min. This procedure was repeated 24 h later, and the animals were sacrificed and macrophages were harvested, as described above, another 24 h later. Isolated alveolar macrophages were stimulated with OX8 (anti-CD8) and examined for TNF and IL-1ß release as described above. RT-PCR and Western blot analysis confirmed that Syk antisense-treated alveolar macrophages did not express Syk mRNA or protein, compared with liposome or scrambled nucleotide + liposome-treated cells.⁴

RT-PCR

Alveolar lavage cells were incubated with OX41-FITC and sorted on a Coulter EPICS Elite cell sorter (Coulter Electronics) to obtain a population with <1% OX52 (T lymphocyte) contamination. Total RNA was extracted using TRIzol reagent (Life Technologies, Burlington, Canada) yielding 3.1 ± 0.4 µg/10⁶ cells with an OD_260/280 ratio of 1.85. mRNA was reverse transcribed by SuperScript RNase (Life Technologies) using a PTC-100 Programmable Thermal Controller (MJ Research, Cambridge, MA) according to the manufacturer’s protocols.

The magnitude of cDNA synthesis was tested using paper chromatography (28). PCR was a modification of the Life Technologies Taq DNA polymerase protocol, with changes in the concentration of dNTPs (1.23 mM) and 10x PCR buffer (67 mM Tris (pH 8.8), 1.5 mM MgCl₂, 16.6 mM (NH₄)₂SO₄, and 10 mM BME) in a total volume of 20 µl. 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 TNF- 5' primer: 5'-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3' and 3' primer: 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3'; 3) rat IL-1ß 5' primer: 5'-GAA GCT GTG GCA GCT ACC TAT GTC T-3' and 3' primer: 5'-CTC TGC TTG AGA GGT GCT GAT GTA C-3'; 4) rat CD8 5' primer: 5'-CAG TTA CAG TTG TCA CCA AA-3' and 3' primer: 5'-CAC GAA TTT CTC TGA AGG TC-3'; 5) rat CD8ß 5' primer: 5'- TTC AGA CTC CTT CAT CCC TG-3', 3' primer: 5'-ACA GTT CGG AAA GAC CCA GA-3'; and 6) rat GAPDH 5' primer: 5'-CTG GTG CTG AGT ATG TCG TG-3' and 3' primer: 5'-CAG TCT TCT GAG TGG CAG TG-3'. The PCR products for ß-actin, TNF-, IL-1ß, CD8, CD8ß, and GAPDH were 526, 354, 520, 630, 428, and 294 bp, respectively. Competitor cDNA (MIMIC) (Clontech Laboratories, Palo Alto, CA) was used to quantitate TNF- message according to the manufacturer’s instruction and generated a modified PCR product for TNF-, 500 bp. After preliminary test of PCR cycle numbers, 21 cycles were used for ß-actin, TNF-, and IL-1ß, (95°C for 45 s, 62°C for 45 s, and 72°C for 2 min), and 30 cycles were used for CD8, CD8ß, and GAPDH (95°C for 45 s, 56°C for 45 s, and 72°C for 2 min). Products were run on a 2% agarose gel and stained with ethidium bromide (EtBr).

Statistical analysis

Data were subjected to analysis of variance with correction for multiple comparisons (Scheffee’s test). Results were considered significantly different when p < 0.05. Throughout the text, data were expressed as mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Macrophages express CD8 mRNA and protein using RT-PCR and flow cytometry analysis

RT-PCR analysis was employed to examine the expression of CD8 mRNA in the alveolar macrophage line NR8383 cells. Consistent with our previous RT-PCR result that freshly isolated alveolar macrophages contained CD8 mRNA (15), NR8383 cells were positive for both CD8 and CD8ß mRNA (Fig. 1a) indicating that macrophages synthesize CD8 molecules. No PCR products were detected when reverse transcriptase was omitted (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 1. RT-PCR and flow-cytometry analysis of CD8 expression by alveolar macrophage cell line NR8383 cells. RNA from NR8383 cells was reverse transcribed and analyzed by PCR reactions with primers for CD8 (630 bp), CD8ß (400 bp), and GADPH (294 bp) (a). NR8383 cells were stained with Ab OX8-FITC (CD8) (b), 341-FITC (c), or mouse IgG1-FITC.

OX8 (CD8) and 341 (CD8) stimulate TNF and IL-1ß production by alveolar macrophages

To examine the effects of CD8 and ß on TNF and IL-1ß production by alveolar macrophages, we incubated alveolar macrophages (1x10⁶ cells/ml) with Abs OX8 (CD8) and 341 (CD8ß) at the dose of 0.5, 2, 5, or 10 µg/ml. Cell-free supernatants were used to determine TNF contents after 6 h incubation and to test IL-1ß contents after 24 h incubation. As shown in Fig. 2, the isotype control IgG1 did not modify TNF and IL-1ß production. Both OX8 and 341 stimulated TNF production by alveolar macrophages in a dose-dependent manner (Fig. 2a). In contrast, OX8 but not 341 dose-dependently stimulated IL-1ß production (Fig. 2b). Interestingly, both OX8 and 341 induced mRNA expression for IL-1ß (Fig. 3). As described for NO production (15), Ab to the N-terminal Ig-like variable region of T cell CD8 (G28) and the isotype control Ab IgG2a did not induce TNF or IL-1ß production by alveolar macrophages (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Effects of OX8 (CD8) and 341 (CD8ß) on TNF and IL-1ß production by alveolar macrophages. Alveolar macrophages (1 x 106 cells/ml) were incubated with OX8, 341, or isotype control IgG1 for 6 or 24 h. Cell-free supernatants were used to determine TNF (6 h) (a) or IL-1ß (24 h) (b). Results are means ± SEM for four to six (a) or three (b) experiments (*, p < 0.05 by comparison with cells treated with corresponding dose of IgG1).

View larger version (99K): [in this window] [in a new window]   FIGURE 3. PCR analysis for TNF and IL-ß of FACS-enriched alveolar macrophages (89% positive for OX41, <1% T lymphocyte (OX52) contamination). Alveolar macrophages were incubated with OX8 (CD8), 341 (CD8ß), or isotype control IgG1 at the dose of 5 µg/ml for 4 h. RNA was extracted and subjected to RT-PCR analysis for (a) TNF and IL-ß and (b) the quantitation of TNF cDNA. MIMIC was used as a competitive cDNA template at the dose of 10 attomole (lane 1), 1 attomole (lane 2), 0.1 attomole (lane 3), 0.01 attomole (lane 4), 0.001 attomole (lane 5), and no MIMIC (lane 6). One of three independent experiments is shown.

View this table: [in this window] [in a new window]   Table I. OX8 (CD8) and 341 (CD8ß) stimulate mediator secretion from NR8383 cells1

To examine whether IFN- is induced in bronchoalveolar lavage (BAL) macrophage cell suspensions after CD8 stimulation and exclude the possibility that NK cells may contribute in macrophage activation, alveolar macrophages were incubated with 5 or 10 µg/ml of OX8 (CD8) or IgG1 for 2, 6, 18, or 24 h. IFN- levels were determined in the cell-free supernatants. IFN- production by alveolar macrophages was not significantly affected by treatment with OX8 for 2 to 24 h (5 µg/ml: 3.3–15.1 pg/10⁶ cells; 10 µg/ml: 0–9.6 pg/10⁶ cells, n = 4) as compared with that by cells treated with IgG1 (10 µg/ml) for 2 to 24 h (1.4–9.0 pg/10⁶ cells, n = 4) or compared with sham-treated groups (0.5–6.9 pg/10⁶ cells) (p > 0.05, n = 4).

To compare the potency of CD8 with that of LPS in mediating TNF and IL-1ß production, alveolar macrophages were stimulated with OX8 (anti-CD8, 10 µg/ml), IgG1 (10 µg/ml) or LPS (1 µg/ml) for 6 or 24 h. Cell-free supernatants were used to determine TNF (6 h) or IL-1ß (24 h) production. OX8-stimulated TNF production (770.9 ± 91.4 pg/10⁶ cells, n = 4) by alveolar macrophages was comparable to that induced by LPS (669.9 ± 69.0 pg/10⁶ cells, n = 4). Spontaneous TNF production (127.5 ± 10.9 pg/10⁶ cells) was not significantly modified by IgG1 (140.1 ± 10.8 pg/10⁶ cells, n = 4). Similarly, IL-1ß production after stimulation with OX8 (43.7 ± 8.6 pg/10⁶ cells, n = 4) was comparable to that induced by LPS (52.9 ± 11.9 pg/10⁶ cells, n = 4). IL-1ß production was not induced by IgG1 (9.3 ± 0.9 pg/10⁶ cells) as compared with no treatment group (13.2 ± 4.3 pg/10⁶ cells) (p > 0.05, n = 4).

OX8 (CD8) and 341 (CD8ß) stimulated mRNA expression for TNF and IL-1ß

To further confirm that CD8 stimulates TNF and IL-1ß production, RT-PCR analysis was used to test mRNA expression for TNF and IL-1ß. FACS-enriched alveolar macrophages (<1% OX52 (T lymphocyte) contamination) were incubated with OX8 (CD8), 341 (CD8ß), or IgG1 at the dose of 5 µg/ml for 4 h. RNA was extracted and subjected to RT-PCR. As shown in Fig. 3a, both OX8 (CD8) and 341 (CD8ß) stimulated mRNA expression for TNF and IL-ß. To further quantitate the amount of TNF generated from OX8- or 341-treated cells, the competitive cDNA template (MIMIC) which produced an altered PCR product (500 bp) was used (Fig. 3b). TNF- mRNA (assuming RNA was fully transcribed into cDNA during the RT reaction) was calculated from the concentration of MIMIC. We showed that alveolar macrophages stimulated by OX8 (CD8) and 341 (CD8ß) produced 311.0 ± 72.2 and 248.4 ± 85.6 attomole TNF mRNA/µg total RNA, respectively.

Inhibition of OX8-induced TNF and IL-1ß production by PP1 and genistein

To examine the role of PTK in CD8-mediated TNF and IL-1ß production, we used the inhibitors PP1 (1, 5, 10, or 20 µg/ml, equivalent to 3.6, 17.8, 35.5, and 71.1 µM, respectively) or genistein (0.5, 1, 10, 50 µg/ml, equivalent to 1.9, 3.7, 37.0, and 185.0 µM, respectively). These concentrations of genistein and PP1 did not affect the viability of the macrophages (trypan blue exclusion). Alveolar macrophages (1x10⁶ cells/ml) were incubated with OX8 or IgG1 (5 µg/ml) for 6 h (TNF) or 24 h (IL-1ß) in the presence or absence of the PTK inhibitors. Cell-free supernatants were used to test TNF and IL-1ß. PP1 (Fig. 4) and genistein (Fig. 5) inhibited both TNF and IL-1ß production in a dose-dependent manner. PP1 at the dose of 10 and 20 µg/ml markedly inhibited OX8-mediated TNF (88.1% and 91.4%) and IL-1ß (88.2% and 100%) production. Similarly, genistein at the dose of 10 and 50 µg/ml significantly blocked OX8-induced TNF (50.4% and 69.1%) and IL-1ß (96.6% and 100%) production. Spontaneous or IgG1-mediated TNF and IL-1ß production were not modified by PP1 or genistein (data not shown). In other experiments, alveolar macrophages were treated with various doses of OX8 (2, 5 or 10 µg/ml) in the presence or absence of genistein (10 µg/ml). We further confirmed that TNF production by various doses of OX8 was inhibited by genistein (10 µg/ml) (p < 0.05, n = 3, data not shown). TNF production by peritoneal macrophages was dose-dependently stimulated by OX8, and inhibited by genistein (82.6% at 20 µg/ml) in a similar manner as that seen in alveolar macrophages (n = 5, data not shown).

View larger version (27K): [in this window] [in a new window]   FIGURE 4. PP1 (a relative specific inhibitor of Src family protein tyrosine kinases) inhibits OX8 (CD8)-induced TNF (a) and IL-1ß (b) production. Alveolar macrophages (1 x 106 cells/ml) were incubated with OX8 (5 µg/ml) in the presence or absence of PP1 (1, 5, 10, 20 µg/ml) for 6 or 24 h. Cell-free supernatants were used to determine TNF (6 h) (a) or IL-1ß (24 h) (b). Results are means ± SEM for three experiments (*, p < 0.05 by comparison with cells treated with OX8 alone; #, p < 0.05 by comparison with cells without treatment).

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Genistein (an inhibitor of protein tyrosine kinases) inhibits OX8 (CD8)-induced TNF (a) and IL-1ß (b) production. Alveolar macrophages (1 x 106 cells/ml) were incubated with OX8 (5 µg/ml) in the presence or absence of genistein (0.5, 1, 10, and 50 µg/ml) for 6 or 24 h. Cell-free supernatants were used to determine TNF (6 h) (a) or IL-1ß (24 h) (b). Results are means ± SEM for three experiments (*, p < 0.05 by comparison with cells treated with OX8 alone; #, p < 0.05 by comparison with cells without treatment).

To further extend our understanding of the involvement of PTK in CD8-mediated macrophage activation, we determined PTK activity in cell lysates from alveolar macrophage line NR8383 cells stimulated with OX8 (CD8) or IgG1. PTK activity was stimulated by OX8 (10 µg/ml) after 5 min incubation. Pretreatment of NR8383 cells with genistein for 30 min completely blocked OX8-induced PTK activation (Fig. 6).

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Activation of protein tyrosine kinase (PTK) activity by OX8 (CD8) in alveolar macrophage line NR8383 cells. Cells (1 x 106 cells/ml) with or without genistein (10 µg/ml) pretreatment for 30 min were washed and stimulated with OX8 (10 µg/ml) or IgG1 (10 µg/ml for 5 min). After lysis with extraction buffer (see Materials and Methods), PTK activity in cell lysates was determined using an ELISA system. Data shown are representative of three independent experiments in triplicate (*, p < 0.05 by comparison with cells treated with IgG1; #, p < 0.05 by comparison with cells without genistein treatment).

Wortmannin, an antagonist of PI-3-kinase (29) and Ro 31-8220, a potent antagonist of PKC (30), were used to examine OX8 (CD8)-mediated signaling. Wortmannin (10, 100, or 1000 nM) or Ro 31-8220 (0.01–10 µM) was incubated with alveolar macrophages (2x10⁶ cells/ml) for 6 h or 24 h in the presence or absence of OX8 (5 µg/ml). Cell-free supernatants were used to test TNF (6 h) or IL-1ß (24 h) release. Wortmannin did not affect OX8-induced TNF production (Fig. 7a), and only at a high dose (1000 nM) partially (42.9%) reduced OX8-induced IL-1ß production (Fig. 7b).

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Effect of wortmannin (an inhibitor of PI 3-kinase) on OX8 (CD8)-induced TNF (a) and IL-1ß (b) production. Alveolar macrophages (1 x 106 cells/ml) were incubated with OX8 (5 µg/ml) in the presence or absence of wortmannin (10, 100, and 1000 nM) for 6 or 24 h. Cell-free supernatants were used to determine TNF (6 h) (a) or IL-1ß (24 h) (b). Results are means ± SEM for three experiments (*, p < 0.05 by comparison with cells treated with OX8 alone; #, p < 0.05 by comparison with cells without treatments).

View larger version (33K): [in this window] [in a new window]   FIGURE 8. Effect of Ro 31-8220 on OX8 (CD8)-induced TNF (a) and IL-1ß (b) production. Alveolar macrophages (1 x 106 cells/ml) were incubated with OX8 (5 µg/ml) in the presence or absence of Ro 31-8220 (0.01, 0.1, 1, 5, or 10 µM) for 6 or 24 h. Cell-free supernatants were used to determine TNF (6 h) (a) or IL-1ß (24 h) (b). Results are means ± SEM for four experiments (*, p < 0.05 by comparison with cells treated with OX8 alone; #, p < 0.05 by comparison with cells without treatments).

Syk antisense inhibition of TNF, but not IL-1ß, release from anti-CD8 (OX8)-stimulated alveolar macrophages

Compared with scrambled oligonucleotide/liposome control, treatment with Syk antisense significantly (p 0.05) down-regulated OX8 (2 µg/ml; 57.3% inhibition, and 5 µg/ml; 85.6% inhibition)-stimulated TNF release (Fig. 9a). In contrast, OX8-stimulated IL-1ß release was not inhibited by Syk antisense (Fig. 9b). Interestingly, in these experiments the levels of TNF, but not IL-1ß, production were approximately 10-fold lower than in other experiments (compare Figs. 4, 5, 7, and 8). Careful analysis showed that this was attributable to the liposome preparation alone and was specific to TNF production. However, this observation did not detract from the significant inhibition of TNF production by the antisense to Syk.

View larger version (28K): [in this window] [in a new window]   FIGURE 9. Syk kinase antisense inhibits OX8 (anti-CD8) induced TNF (a), but not IL-1ß (b), release. Alveolar macrophages were isolated from rats treated with liposome alone, scrambled nucleotide, or Syk kinase antisense, and stimulated with OX8 (2 and 5 µg/ml) or isotype control. Compared with scrambled oligonucleotide, Syk kinase antisense significantly inhibited (p 0.05) OX8 induced TNF release (a). In contrast, Syk antisense did not inhibit OX8-induced IL-1ß release (b). Liposome, scrambled oligonucleotide, and isotype control did not significantly affect TNF (a) or IL-1ß (b) release. Results are expressed as mean ± SEM for three experiments (*, p 0.05, comparison between scrambled oligonucleotide and antisense treated alveolar macrophages stimulated with OX8).

Lack of an effect of OX8 (CD8) and 341 (CD8ß) on PGE₂ production or phagocytosis by alveolar macrophages

To examine the effect of CD8 and -ß on arachidonic acid metabolism, PGE₂ production was tested after incubation of alveolar macrophages with OX8 (CD8) and 341 (CD8ß) for 24 h. PGE₂ production was not affected by OX8 at the dose of 2 µg/ml (173.9 ± 20.3 pg/10⁶ cells), 5 µg/ml (370.6 ± 8.9 pg/10⁶ cells), or 10 µg/ml (449.7 ± 106.4 pg/10⁶ cells) compared with isotype control IgG1 with the corresponding dose of 2 µg/ml (237.9 ± 50.2 pg/10⁶ cells), 5 µg/ml (320.5 ± 30.3 pg/10⁶ cells), or 10 µg/ml (570.9 ± 54.1 pg/10⁶ cells) (n = 3, p > 0.05). Similarly, 341 (CD8ß) at the dose of 2, 5, or 10 µg/ml did not influence PGE₂ production compared with IgG1 (n = 3, data not shown). As a positive control, LPS (1 µg/ml) stimulated PGE₂ production by alveolar macrophages (1502.2 ± 420.6 pg/10⁶ cells) compared with the no treatment group (138.8 ± 32.7 pg/10⁶ cells) (p < 0.05, n = 3). Production of PGE₂ was also elevated by IgG1 (2, 5, or 10 µg/ml) compared with the no treatment group (p < 0.05, n = 3).

Phagocytosis of unopsonized or IgG-opsonized sheep erythrocytes by NR8383 was not altered by treatment of the cell line with anti-CD8 (OX8). Under the conditions we used, opsonization enhanced percentage phagocytosis by at least 5-fold, but OX8 had no effect on this percentage or on the phagocytic index (5–7 erythrocytes/phagocytic cell). These observations were consistent regardless of whether the NR8383 had been concurrently treated with OX8, or pretreated for 8 or 20 h in an effort to modify their phagocytic phenotype.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, we identified that rat alveolar and peritoneal macrophages express novel CD8 molecules on their surface (15). Given the concerns of interpretation of RT-PCR results due to the purity of alveolar macrophage populations (15), we used a macrophage cell line NR 8383 cell to analyze CD8 message and protein expression. We demonstrated that both mRNA and proteins for CD8 were expressed by NR 8383 cells. These data further confirm that macrophages synthesize CD8.

One of the important questions that remains to be addressed is the biological significance of CD8 on macrophages. To approach this, we focused on two major macrophage-derived cytokines, TNF and IL-1ß. We demonstrated for the first time that both CD8 (OX8) and CD8ß (341) significantly stimulate the mRNA or protein production for TNF (Figs. 2 and 3). TNF production was specific to CD8 stimulation (not due to cross-linking any surface molecules) because an Ab to another cell surface marker on alveolar macrophages (OX41, data not shown) did not induce TNF secretion. Interestingly, TNF production mediated by OX8 (CD8) at a dose of 10 µg/ml (1130.5 ± 105.9 pg/10⁶ cells) was comparable to that induced by LPS (10 ng/ml) + IFN- (500 U/ml) (1610.3 ± 119.9 pg/10⁶ cells), in comparison with IgG1 or no treatment groups (78.9 ± 26.2 and 62.9 ± 18.5 pg/10⁶ cells, respectively). There is considerable evidence that macrophage-derived TNF is important in host defense against various pathogens including P. carinii (4), Leishmania major (31), Listeria monocytogenes (32), etc. Indeed, our recent data indicate that OX8 (CD8) (5 and 10 µg/ml) stimulation of alveolar macrophages significantly (p < 0.01) decreases the proportion of these cells infected with L. major (33). The crucial roles of IL-1ß and NO in defense, or infection with influenza or other pathogens, have been demonstrated using IL-1ß-deficient animal models or by disruption of NO synthesis (34, 35, 36). Our data on the effects of CD8 on the stimulation of TNF, IL-1ß, and NO production by macrophages, together with the anti-L. major effects, suggest that macrophage CD8 may have profound effects in host defense against infections.

However, depending upon the disease model, the effect of TNF and IL-1ß may be either beneficial or harmful (8). The significant proinflammatory effects of TNF and IL-1ß have been well documented in a number of settings, such as LPS-induced lethal toxicity (7). Thus, the elucidation of the effects of CD8 in macrophage-implicated inflammatory reactions may provide new insights in understanding the roles of macrophages in inflammation.

CD8ß has been shown to be involved in thymic lymphocyte selection (37). However, the role of CD8ß in mature T lymphocytes is unclear, since CD8ß gene-targeted mice (lacking CD8ß) appear to have normal CD8 effector function (38). Recently, studies have demonstrated that CD8ß is required for the recognition of peptide ligands (39), increases CD8 coreceptor function (40), and influences CD8-associated p56^lck kinase activity (41). However, stimulation of T lymphocyte function by CD8ß independent of CD8 has not been reported. Interestingly, our study indicated that activation of CD8ß by Ab 341, independent of CD8 stimulation, significantly promotes TNF and NO production, and mRNA expression for TNF and IL-1ß by alveolar macrophages or a macrophage cell line NR 8383 cells (Figs. 2 and 3 and Table I). These results, together with our flow cytometry data (antigenic differences within the ligand-binding domain of CD8 between macrophages and T cells) (15), demonstrate that CD8 on macrophages may be functionally and structurally distinct from CD8 on T cells.

341 (CD8ß) stimulated IL-1ß secretion by NR8383 cells (Table I). However, in alveolar macrophages, 341 did not affect IL-1ß secretion (Fig. 2b), although it stimulated the mRNA expression for IL-1ß (Fig. 3). The mechanisms of this discrepancy of the effect of 341 in NR8383 cells vs that in alveolar macrophages are unclear. Distinct mechanisms employed by different cell types in the regulation of IL-1ß secretion may be relevant.

Interestingly, activation of CD8 on macrophages induces selective mediator secretion (TNF, IL-1ß, and NO) rather than overall stimulation of macrophage function. IFN- and PGE₂ production by macrophages is not elevated by OX8 (CD8) compared with the corresponding dose of IgG1 isotype control Ab. In contrast, LPS dramatically stimulates PGE₂ production by alveolar macrophages. IgG1 (2, 5, or 10 µg/ml) elevates PGE₂ secretion, probably through the activation of Fc receptors as has been shown by other investigators (42). Our observations that percentage phagocytosis and phagocytic index for unopsonized and opsonized erythrocytes are not altered by activation of macrophage CD8 confirms earlier work on fluorescent microspheres and L. major (our unpublished observations).

To examine CD8-induced intracellular signal transduction pathways involved in the stimulation of macrophage function, we used several inhibitors (see Fig. 10): e.g., PP1 and genistein to block PTK (43, 44), wortmannin to inhibit PI-3 kinase (29), and Ro 31-8220 to antagonize PKC (30). We demonstrated that both TNF and IL-1ß production were significantly inhibited by PP1 and genistein. Moreover, OX8 stimulated PTK activity in macrophages. These data suggest that PTKs are involved in the CD8-mediated effects. It is well documented in T cells that CD8 is functionally and physically associated with p56^lck (45). Although there is no report of p56^lck expression by macrophages, other Src family tyrosine kinases, such as p59^fyn, p53/56^lyn, p58^hck, and p59^fgr are expressed by macrophages (46, 47, 48, 49, 50). Most of these Src family kinases are involved in LPS-induced TNF or IL-1ß production by macrophages (47, 48, 49). Moreover, function of p56^lck can be substituted by other Src family kinases such as p59^fyn (51). Given the selective effect of PP1 on src family tyrosine kinases (43), candidates involved in CD8-mediated TNF and IL-1ß production may include such tyrosine kinases as p59^fyn, p53/56^lyn, p58^hck, and p59^fgr. Immunoprecipitation experiments may help to clarify the roles of these tyrosine kinases in CD8-mediated signaling in macrophages.

View larger version (17K): [in this window] [in a new window]   FIGURE 10. Differential effects of kinase inhibitors on CD8-mediated TNF and IL-1ß production.

Recent advances in understanding the role of PI 3-kinase as an important element in signaling pathways have largely been made possible by the availability of inhibitors of the enzyme, in particular the fungal metabolite wortmannin (52). In monocytes/macrophages, PI 3-kinase is believed to be a downstream effector of tyrosine kinases after LPS stimulation (52). To investigate the role(s) of PI 3-kinase in CD8-mediated macrophage activation, wortmannin was used in our study. Interestingly, wortmannin did not affect OX8 (CD8)-induced TNF production (Fig. 7a), and at a larger dose (1 µM) partially reduced IL-1ß production (Fig. 7b). Given that higher concentrations (>1 µM) of wortmannin have been shown to inhibit other enzymes such as PI 4-kinase, phospholipase A₂, or myosin light chain kinase (53), the mechanism of this partial inhibition is unclear. These data, together with the lack of effect of wortmannin on OX8-mediated NO production, suggest that PI 3-kinase may not be an important element in CD8-mediated signaling in macrophages.

It has recently been shown that Syk kinase plays a differential role in macrophage signaling. Like PI-3 kinase, Syk kinase has been implicated in Fc receptor signal transduction, but may not play a role in LPS-mediated TNF and IL-1ß release (17, 18). However, these studies used high doses of LPS, which may act in a CD14-independent manner (54). Using a low dose of LPS (10 ng/ml), we demonstrated that LPS stimulation of TNF release is at least in part Syk dependent.⁴ As our results show that PI-3 kinase is not involved in CD8-mediated stimulation of TNF, we investigated the upstream Syk kinase signaling event to determine whether it is involved in CD8-mediated stimulation of alveolar macrophages. Syk antisense inhibited TNF but not IL-1ß release from OX8 (CD8) stimulated alveolar macrophages (Figs. 9 and 10). Therefore, CD8-mediated macrophage signaling appears to function through multiple pathways. TNF and IL-1ß are Src-kinase dependent, however, TNF is Syk dependent but PKC independent, whereas IL-1ß is Syk independent but PKC dependent. There is some similarity with T lymphocyte signaling, in which CD8 associated p56^lck allows Zap 70 (Syk homologue) to interact with the TCR to mediate cell signaling (16). However, unlike our current understanding for T lymphocytes, there appear to be Syk (ZAP 70)-independent pathways of CD8-mediated stimulation in macrophages.

Considerable evidence indicates that monocytes/macrophages possess mechanisms that regulate TNF and IL-1ß production differently (12, 55, 56). Interestingly, Ro 31-8220 showed disparate effects on OX8 (CD8)-mediated TNF and IL-1ß production by macrophages. Ro 31-8220 completely abolished OX8-mediated IL-1ß production by macrophages (Fig. 8b), but did not affect OX8-induced TNF (Fig. 8a), suggesting distinct mechanisms involved in CD8-mediated TNF and IL-1ß production.

In addition to defining the kinases involved, it is essential to determine whether CD8 acts independently, or as a coreceptor, in mediating macrophage stimulation. We used intact anti-CD8 and ß Abs to stimulate macrophage function. These Abs could bind Fc receptors in turn stimulating macrophage activities. Alveolar macrophages express all three Fc receptors, FcRI (CD64), FcRII (CD32), and FcRIII (CD16) (57). FcRI binds monomeric IgG with high affinity, FcRII and FcRIII bind monomeric IgG with low affinity, but bind aggregated IgG (58). Two important kinases in the signaling cascades of FcRI (59, 60), FcRII (61), and FcRIII (27, 62) are PI3-kinase and Syk kinase.

In our system, OX8-induced NO, TNF, and IL-1ß release are PI3-kinase independent. Additionally, OX8-mediated IL-1ß release is also Syk kinase independent. These results, together with the lack of effect of isotype control reagents and lack of effect on IgG-opsonized phagocytosis, suggest that CD8-induced macrophage stimulation for NO, TNF, and IL-1ß production are Fc receptor independent. However, IgG1 stimulated PGE₂ release at similar levels to anti-CD8 (OX8), suggesting that, for OX8-induced PGE₂ stimulation, Fc receptors may be involved. A more detailed analysis of the potential involvement of Fc receptors in CD8-mediated activation of macrophages is required.

In summary, we have demonstrated that macrophages express CD8 mRNA. Activation of CD8 (OX8) or CD8ß (341) significantly stimulated mRNA or protein expression for TNF or IL-1ß by alveolar macrophages or the macrophage line, NR8383 cells. These data, together with the stimulation of NO production (15), indicate that CD8 on macrophages has a significant role in the regulation of macrophage function. Moreover, PTK inhibitors PP1 and genistein inhibited both CD8 (OX8)- and CD8ß (341)-induced TNF and IL-1ß production. In contrast, Ro 31-8220 (PKC inhibitor) and Syk antisense selectively modulated OX8-induced TNF and IL-1ß production. Thus, there are multiple CD8-induced macrophage activation pathways that may involve PTKs, specifically Src-kinases, and differentially regulated pathways, including Syk kinase and PKC, that distinctly modulate TNF and IL-1ß production.

	Acknowledgments

 
We thank Mrs. Fran Thompson for secretarial support, and Drs. Elyse Bissonnette and Redwan Moqbel for advice and helpful comments.

	Footnotes

 
¹ This work was supported by Alberta Lung Association and Medical Research Council of Canada grants to A.D.B. and by National Institutes of Health Grant AI-22193.

² T.-J.L., N.H., and G.R.S. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. A. Dean Befus, Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. E-mail address:

⁴ Abbreviations used in this paper: PKC, protein kinase C; PI, phosphatidylinositol; PTK, protein tyrosine kinase.

⁵ G. R. Stenton, M. K. Kim, P. Hwang, J. G. Park, O. Nohara, N. Hirji, F. L. Wills, M. Gilchrist, W. Finlay, R. L. Jones, A. D. Befus, and A. D. Schreiber. Aerosolized Syk antisense suppresses syk expression, mediator release from alveolar macrophages and pulmonary inflammation. Submitted for publication.

Received for publication March 29, 1999. Accepted for publication December 3, 1999.

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