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The Journal of Immunology, 1998, 160: 6004-6011.
Copyright © 1998 by The American Association of Immunologists

Mechanisms of Macrophage Stimulation Through CD8: Macrophage CD8{alpha} and CD8ß Induce Nitric Oxide Production and Associated Killing of the Parasite Leishmania major1

Nadir Hirji*, Tong-Jun Lin*, Elyse Bissonnette*, Miodrag Belosevic{dagger} and A. Dean Befus2,*

Departments of * Medicine and {dagger} Biological Sciences, University of Alberta, Edmonton, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8{alpha} (OX8, 5 µg/ml) or CD8ß (341, 10 µg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8{alpha} and CD8ß stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 µg/ml), PP1 (src family kinase inhibitor, 5 µg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 µg/ml), and Ro 31-8220 (PKC inhibitor, 1 µM) significantly inhibited anti-CD8{alpha}- and anti-CD8ß-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8{alpha} stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the ß-chain of macrophage CD8, in addition to the {alpha}-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO)3 is an important mediator synthesized from the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS). NO has been implicated in a variety of biologic functions, including neurotransmission, host defense, bronchodilation, and tumor cytotoxicity (1, 2). When macrophages are stimulated with LPS or IFN-{gamma}, they produce copious amounts of NO, due to the up-regulation of inducible NOS (iNOS) (1, 2). In addition to paracrine and juxtacrine effects, macrophage NO also has an autocrine effect. The NO inhibitor, N(G)-monomethyl-L-arginine (L-NMMA) increases macrophage production of IL-1ß and IL-6 (3).

CD8 is a cell surface glycoprotein best known for its expression on a subset of T lymphocytes with suppressor/cytotoxic functions and on NK cells (4, 5). We have shown recently that rat macrophages contain both the message and protein for CD8 (6). Macrophage CD8 is a heterodimer composed of an {alpha}- and ß-chain, and differs from T lymphocyte CD8 within the ligand binding domain of the {alpha}-chain (6).

Our initial studies on the function of macrophage CD8 determined that cross-linking the {alpha}-chain, with the Ab OX8 (anti-CD8{alpha}), stimulated the release of NO (6). However, there is no understanding of the role of ß-chain in macrophage stimulation. On T lymphocytes, the ß-chain of CD8 does not appear to play a role in T lymphocyte effector function, but may be important in their maturation (7). Because we have evidence for differences, both in structure and function, between macrophage and T lymphocyte CD8, we investigated the role of CD8ß on macrophage effector function (NO production).

To further understand the function of macrophage CD8, the pathway(s) involved in CD8{alpha} and/or CD8ß stimulation of NO production was examined. As CD8{alpha} is linked to the protein tyrosine kinase p56lck in T lymphocytes (8, 9), and there is evidence that protein tyrosine kinase activity is involved in macrophage NO production (10, 11), we used the broad spectrum protein tyrosine kinase inhibitor genistein (12) and an inhibitor of the src family kinases, PP1 (13), to elucidate part of the pathway(s) involved in CD8-stimulated NO production. In addition to tyrosine kinase activity, we examined the roles of protein kinase C (PKC), inhibitors polymyxin B (14) and Ro 31-8220 (15), and PI3 kinase, inhibitor wortmannin (16), on CD8{alpha} (OX8)- and CD8ß (341)-stimulated NO production.

As the production of macrophage NO is dependent on the enzyme iNOS, we examined whether cross-linking CD8{alpha} and/or CD8ß up-regulated the production of iNOS protein in alveolar macrophages. Moreover, the tyrosine kinase inhibitors, genistein and PP1, the PKC inhibitors, polymyxin B and Ro 31-8220, and the PI3 kinase inhibitor, wortmannin, were used to elucidate the mechanisms of CD8-stimulated iNOS production.

Finally, given the important role of macrophages in host defense, we examined whether CD8 could affect macrophage cytotoxicity. We used Leishmania major, an intracellular parasite that causes human disease ranging from self-healing ulceration to fatal systemic infection (17), as a model to test macrophage host defense. During the life cycle of Leishmania, the parasite replicates within macrophages (17). Therefore, we examined whether infected macrophages could be induced to kill this parasite when stimulated with either anti-CD8{alpha} (OX8) or anti-CD8ß (341).


   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 (St. Constant, Quebec) and maintained in an isolated room in filter-top cages to minimize unwanted infections. The animals were given food and water ad libitum and maintained on a 12-h (0700 h) to 12-h (1900 h) light-dark cycle. No experimental procedures were performed on animals within the first week after arrival, decreasing the effects of stress associated with transport, new housing facilities, and handling. All experimental procedures were approved by University of Alberta Animal Care Committee (Edmonton, Alberta) in accordance with the guidelines of Canadian Council on Animal Care.

Isolation of bronchoalveolar cells

Animals were anesthetized by i.p. injection of 0.5 ml of Rompun (xylazine) and 0.5 ml of Ketalean (ketamine). The trachea was catheterized with a polyethylene tube, and 12 x 5 ml of cold PBS was massaged into the lungs. Lavage cells were pelleted at 200 x g for 20 min and resuspended in PBS (6, 18). Within rat alveolar lavage, the percentage of alveolar macrophages, tested by esterase staining, was 96 ± 0.4% (82.2 ± 1.2 OX41 positive). The expression of CD8 on alveolar macrophages, using an enriched population (89 ± 1% OX41 positive), was 63 ± 5% for CD8{alpha} (Ab OX8) and 52 ± 3% for CD8ß (Ab 341) (6).

Measurement of NO production

Alveolar macrophages were incubated (2 x 105 cells/test) with 0.5 to 10 µg/ml of Ab for 24 h. Cell-free supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% N-(l-naphthyl)ethylenediamine dihydrochloride, 2.5% H3PO4) and incubated for 10 min at room temperature (19). Concentration of NO2- was determined by measuring the absorbance at 540 {eta}m with a Molecular Devices Vmax Kinetic Microplate Reader (Menlo Park, CA). NaNO2 was used as a standard. In experiments with genistein (Calbiochem, La Jolla, CA), PP1 (Calbiochem), wortmannin (Sigma, St. Louis, MO), polymyxin B (Sigma), and Ro 31-8220 (ROCHE, Welwyn Garden City, U.K.), inhibitors were added 10 min before addition of Abs.

Western blot analysis of iNOS protein

Alveolar macrophages (1 x 106 cells/ml) were incubated (24 h) with Abs, OX8 (anti-CD8{alpha}, 0.5–10 µg/ml), 341 (anti-CD8ß, 0.5–10 µg/ml), and IgG1 (isotype control, 0.5–10 µg/ml), and separated on 8% SDS-PAGE, and the proteins were blotted onto Hybond-C Super membrane (Amersham Life Science, Oakville, Ontario, Canada). The membrane was blocked overnight in 10% instant skim-milk powder before incubation with Abs. The membrane was incubated with rabbit anti-murine iNOS (kindly provided by Dr. J. Weidner, Merck Research Laboratories, Rahway, NJ) for 1 h, after which F(ab')2 goat anti-rabbit horseradish peroxidase (The Jackson Laboratory, Bar Harbor, ME) was used to identify specific proteins. Control experiments were run using secondary Ab alone. In addition, experiments with the peptide used to raise the rabbit anti-murine iNOS Ab (peptide NO17, kindly provided by Dr. J. Weidner, Merck Research Laboratories) were used to determine the specificity of the proteins identified by Western blot analysis. Anti-iNOS was preincubated for 1 h with 1 µM of peptide before addition to membrane. Prestained rainbow m.w. standards (Bio-Rad, Mississauga, Ontario, Canada) were used as markers. Visualization of the horseradish peroxidase was done using Western blot chemiluminescence reagent (DuPont, Boston, MA), according to manufacturer’s protocol.

In experiments with genistein (12) (Calbiochem, La Jolla, CA), PP1 (13) (Calbiochem), wortmannin (16) (Sigma), polymyxin B (14) (Sigma), and Ro 31-8220 (15) (ROCHE), inhibitors were added 10 min before addition of anti-CD8{alpha} or anti-CD8ß.

Densitometry of the iNOS protein was determined using ImageMaster 1D/2D gel analysis system (Pharmacia Biotech, Baie D’Urfe, Quebec, Canada).

L. major parasite

The National Institutes of Health 173 (WHOM/Ir/-/173) strain of L. major, isolated from a patient in Iran (20), was used in this study. The amastigotes of L. major were maintained by serial passage in BALB/c mice (50 µl containing 2 x 106 amastigotes inoculated s.c. into footpads) (21, 22). Four to five weeks after infection, monodispersed amastigotes were obtained by disruption of infected footpad tissue and passage through number 50 stainless steel mesh screens into DMEM (Life Technologies, Grand Island, NY) containing 10% FBS (Life Technologies) and gentamicin (Sigma). Parasites were stained with fluorescein diacetate (ICN, Cleveland, OH) and ethidium bromide (ICN) (23), and the number of viable amastigotes was determined using a hemocytometer.

Intracellular parasite-killing assay

Rat alveolar macrophages were suspended in DMEM at a concentration of 1 x 105 cells/0.5 ml in polypropylene tubes (Falcon; Becton Dickinson, Lincoln Park, NJ). To each culture, 4 x 105 viable amastigotes were added and the cultures were incubated for 2 h at 37°C and 5% CO2. Cells were washed twice (0.5 ml DMEM, 200 x g for 5 min) and resuspended in 0.5 ml DMEM. Appropriate concentrations of test solutions were added (0.1 ml), and the cells were incubated for 72 h at 37°C in 5% CO2. After incubation, 0.1 ml of the cell suspension was used to prepare cell smears using a cytocentrifuge (Shandon, Pittsburgh, PA) and stained with Wright stain (Leukostat; Fisher Scientific, Orangeburg, NY). The percentage of infected cells of 200 macrophages was determined by microscopic examination of stained cell smears under oil immersion (21, 23). Experiments were conducted in quadruplicate sets, and repeated three times. The data shown are from one representative experiment.

Statistical analysis

Statistical significance between any two groups was analyzed by two-tailed Student’s t test. Data in text represent mean ± SEM; n = separate experiments (mean of triplicate samples) using pooled cells from two to six rats.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-CD8{alpha} and anti-CD8ß stimulated NO production in alveolar macrophages

The production of NO (24 h) by alveolar macrophages was examined using the Griess reagent (Fig. 1Go). OX8 (anti-CD8{alpha}, 5 µg/ml) and 341 (anti-CD8ß, 10 µg/ml) significantly (p <= 0.05) stimulated the production of NO, compared with IgG1 (10 µg/ml) isotype controls. In addition, an Ab to another surface marker on alveolar macrophages (OX41, 10 µg/ml) did not stimulate NO production.



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  		FIGURE 1. Anti-CD8{alpha} (OX8) and anti-CD8ß (341) stimulated NO release from alveolar macrophages. The release of NO (using Griess reagent) from alveolar macrophages stimulated (24 h) with isotype control (IgG1, 10 µg/ml), anti-CD8{alpha} (OX8, 5 µg/ml), anti-CD8ß (341, 10 µg/ml), or OX41 (granulocyte/alveolar macrophage marker, 10 µg/ml). Concentration of NO2- (µM/106 cells) is represented by mean ± SEM. *p <= 0.05, OX8 and 341 vs IgG1, n = 3–4.

 
As macrophages are sensitive to LPS stimulation, Abs were tested for endotoxin contamination (Limulus E-Toxate test; Sigma). OX8 and 341 were negative for endotoxin (data not shown). As the lower limit of the test is 0.05 to 0.1 endotoxin units (EU) ({approx}0.02–0.04 ng/ml LPS) and with previous work on macrophages demonstrating that 1 ng/ml is not sufficient to induce significant NO release (24), OX8 and 341 stimulation of NO was not due to LPS contamination.

Anti-CD8{alpha} and anti-CD8ß stimulate the production of alveolar macrophage iNOS protein

Because stimulating alveolar macrophages with either anti-CD8{alpha} or anti-CD8ß induced NO production (Fig. 1Go), we examined whether there was an increase in iNOS protein using Western blot. Alveolar macrophages were stimulated with OX8 (anti-CD8{alpha}, 5 µg/ml) or 341 (anti-CD8ß, 10 µg/ml) (24 h) and examined for the up-regulation of iNOS protein (Fig. 2Go). Compared with untreated cells or IgG1 (isotype control, 10 µg/ml)-stimulated cells, OX8 and 341 up-regulated iNOS protein (Fig. 2GoA). Using densitometry (Fig. 2GoC), it was determined that both OX8 and 341 significantly (p <= 0.01, p <= 0.05) stimulated the production of iNOS protein compared with isotype control.



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  		FIGURE 2. Anti-CD8{alpha} and anti-CD8ß stimulated up-regulation of iNOS protein from alveolar macrophages. Western blot analysis of anti-CD8{alpha} (OX8, 5 µg/ml) or anti-CD8ß (341, 10 µg/ml) stimulated (24 h) iNOS production from alveolar macrophages. A, Lane 1, m.w. standards; lane 2, no treatment; lane 3, isotype control (IgG1, 10 µg/ml); lane 4, OX8 (5 µg/ml); and lane 5, 341 (10 µg/ml). B, Anti-murine iNOS was preincubated (1 h) with NO17 (iNOS peptide, 1 µM) before use in Western blot analysis. Lane 1, m.w. standards; lane 2, no treatment; lane 3, isotype control (IgG1, 10 µg/ml); lane 4, OX8 (5 µg/ml); and lane 5, 341 (10 µg/ml). C, Densitometry of iNOS production by anti-CD8{alpha} or anti-CD8ß. **p <= 0.01, OX8 vs IgG1; *p <= 0.05, 341 vs IgG1, n = 3.

 
To determine whether the 130-kDa band was iNOS, the anti-iNOS Ab was preincubated (1 h) with NO17 peptide (1 µM), which corresponds to the antigenic determinant identified by the anti-murine iNOS Ab, before use in Western blot analysis (Fig. 2GoB). The band corresponding to iNOS protein was not identified, indicating that the 130-kDa protein up-regulated by OX8 and 341 is iNOS.

Determination of the mechanisms involved in CD8{alpha}- and CD8ß-stimulated NO production and iNOS up-regulation

Because CD8 in T cells is associated with a protein tyrosine kinase (8, 9) and there is evidence that NO production in macrophages is linked to protein tyrosine kinase activity (10, 11), we examined the effects of a broad range protein tyrosine kinase inhibitor, genistein, on OX8 (5 µg/ml)- and 341 (10 µg/ml)-stimulated NO production by alveolar macrophages (Fig. 3Go). To further elucidate the pathway(s) of CD8 stimulation, inhibitors to PI3 kinase (wortmannin) and PKC (polymyxin B) were also used. Dose-response studies were used to identify optimal concentrations for each inhibitor (data not shown). Genistein (10 µg/ml) and polymyxin B (100 µg/ml) significantly (p <= 0.01) inhibited (24 h) OX8 (5 µg/ml)- and 341 (10 µg/ml)-induced NO production (Fig. 3Go). As the Abs used were shown to be LPS negative, the effects of polymyxin B on OX8- and 341-stimulated NO release cannot be due to polymyxin B binding contaminating LPS in the Ab preparations. In addition, as polymyxin B has been shown to disrupt calmodulin-sensitive processes (25), a more selective inhibitor for PKC, Ro 31-8220 (15), was used to further examine the role of PKC in OX8-stimulated NO production. Ro 31-8220 (1 µM) inhibited (p <= 0.01) OX8 (5 µg/ml)-stimulated NO production (Fig. 3Go). In contrast, wortmannin (PI3 kinase inhibitor, 1 µM) did not inhibit OX8 (5 µg/ml)- and 341 (10 µg/ml)-stimulated NO production (Fig. 3Go). These results suggest that stimulating macrophages through the {alpha} (OX8)- and/or ß (341)-chain(s) of CD8 signals NO production through a protein tyrosine kinase and/or PKC-dependent pathway.



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  		FIGURE 3. Inhibition of anti-CD8{alpha} and anti-CD8ß stimulated NO production by genistein, polymyxin B, and Ro 31-8220. Determination of the mechanisms of anti-CD8{alpha} (OX8, 5 µg/ml) and anti-CD8ß (341, 10 µg/ml) stimulated NO production (Griess reagent, NO2- µM/106 cells) using the inhibitors genistein (broad spectrum protein tyrosine kinase inhibitor, 10 µg/ml), wortmannin (PI3 kinase inhibitor, 1000 nM), polymyxin B (PKC inhibitor, 100 µg/ml), and Ro 31-8220 (1 µM). **p <= 0.01, OX8 vs OX8 + genistein or OX8 + polymyxin B; ##p <= 0.01, 341 vs 341 + genistein or 341 + polymyxin B, n = 3–4.

 
To further elucidate the pathways of macrophage CD8 stimulation, we used Western blot analysis to determine whether genistein, wortmannin, or polymyxin B inhibited OX8 (Fig. 4GoA)- and/or 341 (Fig. 4GoC)-stimulated iNOS production. Genistein (10 µg/ml) and polymyxin B (100 µg/ml) inhibited OX8 (5 µg/ml) and 341 (10 µg/ml) up-regulation (24 h) of iNOS (Fig. 4Go, A and C). Consistent with the NO data in Figure 3Go, wortmannin (1 µM) did not inhibit OX8- or 341-stimulated (24 h) iNOS production (Fig. 4Go, A and C).



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  		FIGURE 4. Inhibition of anti-CD8{alpha} and anti-CD8ß stimulated iNOS production by genistein and polymyxin B. Western blot analysis of the mechanisms of anti-CD8{alpha} (OX8, 5 µg/ml) and anti-CD8ß (341, 10 µg/ml) stimulated iNOS production. Lane 1, m.w. markers; lane 2, anti-CD8{alpha} (A) or anti-CD8ß (C); lane 3, Ab + genistein (broad spectrum protein tyrosine kinase inhibitor, 10 µg/ml); lane 4, Ab + wortmannin (PI3 kinase inhibitor, 1000 nM); and lane 5, Ab + polymyxin B (PKC inhibitor, 100 µg/ml). B (anti-CD8{alpha}) and D (anti-CD8ß), Densitometry of iNOS production by anti-CD8{alpha} or anti-CD8ß + inhibitors, n = 3.

 
The magnitude of the inhibition of iNOS up-regulation was determined by densitometry (Fig. 4Go, B and D). Consistent with the NO data in Figure 3Go, densitometry of iNOS (Fig. 4Go, B and D) confirmed that genistein and polymyxin B inhibited the up-regulation of OX8 (genistein, 48% and polymyxin B, 47% inhibition)- and 341 (genistein, 33% and polymyxin B, 33% inhibition)-stimulated iNOS.

src family kinase(s) is involved in CD8-stimulated NO production and iNOS up-regulation

Data in Figures 3Go and 4Go determined that protein tyrosine kinase(s) is involved in CD8{alpha}- and CD8ß-stimulated NO production and iNOS up-regulation, but did not identify the family of these kinases. Because src family kinases are involved in T lymphocyte CD8 stimulation (8, 9) and are up-regulated in LPS- and IFN-{gamma}-stimulated macrophages (26, 27), we examined the role of src family kinase(s) in CD8{alpha}- and CD8ß-mediated stimulation. PP1 (5, 10, and 20 µg/ml), an inhibitor of src family kinases (13), dose dependently inhibited (p <= 0.01) OX8 (5 µg/ml)- and 341 (10 µg/ml)-mediated NO production (Fig. 5Go).



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  		FIGURE 5. src family kinase(s) is involved in CD8{alpha}- and CD8ß-stimulated NO production. Inhibition of src family kinases (using the inhibitor PP1) to assess their role in anti-CD8{alpha} (OX8, 5 µg/ml)- and anti-CD8ß (341, 10 µg/ml)-stimulated NO production. **p <= 0.01, OX8 vs OX8 + PP1; ##p <= 0.01, 341 vs 341 + PP1, n = 3.

 
To further explore the role of src family kinase(s) in CD8-mediated stimulation, the effects of PP1 on OX8 and 341 up-regulation of iNOS were examined using Western blot analysis (Fig. 6Go). Similar to the results in Figure 5Go, PP1 dose dependently inhibited OX8 (Fig. 6GoA)- and 341 (Fig. 6GoC)-stimulated iNOS production. The magnitude of the inhibition was determined using densitometry. Figure 6Go, B (OX8) and D (341), confirms the dose-dependent inhibition of anti-CD8{alpha} (OX8)- and anti-CD8ß (341)-mediated iNOS production by PP1.



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  		FIGURE 6. src family kinase(s) is involved in CD8{alpha}- and CD8ß-stimulated iNOS production. Western blot analysis of anti-CD8{alpha} (OX8, 5 µg/ml)- and anti-CD8ß (341, 10 µg/ml)-stimulated iNOS production using the src family kinase inhibitor PP1. Lane 1, m.w. markers; lane 2, anti-CD8{alpha} (A) or anti-CD8ß (C); lane 3, Ab + 1 µg/ml PP1; lane 4, Ab + 5 µg/ml PP1; lane 5, Ab + 10 µg/ml PP1; and lane 6, Ab + 20 µg/ml PP1. B (anti-CD8{alpha}) and D (anti-CD8ß), Densitometry of iNOS production by anti-CD8{alpha} or anti-CD8ß + PP1, n = 3.

 
Enhanced antiparasitic activity by anti-CD8 stimulated alveolar macrophages

The functional significance of alveolar macrophage CD8 was further examined using the protozoan parasite L. major. Alveolar macrophages infected with L. major were stimulated for 72 h with either IFN-{gamma} (control), OX8 (anti-CD8{alpha}), 341 (anti-CD8ß), or IgG1 (isotype control), and the differences in infection were examined (Table IGo). After 72 h, 82.3 ± 4.4% of unstimulated alveolar macrophages were infected with the parasite. IFN-{gamma} (200 U/ml) significantly (p < 0.001) decreased the percentage of parasite-infected alveolar macrophages, which correlated with an up-regulation in NO production, compared with unstimulated cells. OX8 dose dependently (0.5 µg/ml, p <= 0.05; 2 µg/ml, p <= 0.01; 5 and 10 µg/ml, p <= 0.001) decreased the number of infected cells, compared with IgG1 isotype controls. In addition, there was a corresponding dose-dependent increase in NO production. In this experiment, 341 (anti-CD8ß) stimulated killing of Leishmania. However, this appeared to be independent of dose and NO production. Further experimentation using 341 failed to initiate a cytotoxic response in Leishmania-infected macrophages. Therefore, CD8ß appears to play a minor or limited role in stimulating macrophage cytotoxicity to Leishmania.


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  		Table I. Cross-linking CD8{alpha} stimulates antiparasitic activity in alveolar macrophages, which is NO dependent1

 
To determine whether the protective effects of OX8 (anti-CD8{alpha}) were NO dependent, aminoguanidine (a NO inhibitor) was used. Amino guanidine (1 mM) reversed the protective effects of IFN-{gamma} (63.5 ± 11.5% infected cells), which was used as the internal control to verify activity. Amino guanidine also reversed OX8 (10 µg/ml, 78 ± 4.1% infected cells)-stimulated antiparasitic activity, suggesting that the protective effects of OX8 (anti-CD8{alpha}) are NO dependent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression of CD8 is not exclusive to CTL. Dendritic cells and macrophages also express CD8 (6, 28, 29). CD8 plays an important role in CTL adhesion and function (5, 30). Previous work on alveolar macrophages demonstrated differences within the ligand binding domain (the {alpha}-chain of CD8) of macrophage CD8 compared with T lymphocytes (6). Initial work on the function of alveolar macrophage CD8 demonstrated that cross-linking CD8{alpha} stimulated NO production from alveolar macrophages (6).

Further evidence for differences between alveolar macrophage and T lymphocyte CD8 was demonstrated by experiments on CD8ß. The Ab 341 (anti-CD8ß) stimulated alveolar macrophage NO production and iNOS up-regulation (Figs. 1Go and 2Go). In contrast, experiments on T lymphocyte CD8ß have demonstrated that this chain plays a role in T lymphocyte maturation. CD8ß knockout mice and transgenic mice expressing tailless CD8ß have decreased numbers of functionally active CD8 positive cells (31, 32). Additional work has identified a role for CD8ß in recognition of altered peptide ligands (33). These studies identify a role for T lymphocyte CD8ß in maturation and ligand binding. Our work demonstrates that CD8ß is able to regulate, either directly or indirectly, macrophage effector function by stimulating mediator release.

The mechanisms of CD8{alpha}- and CD8ß-stimulated macrophage function were examined using inhibitors to different signaling pathways. In T lymphocytes, CD8 is associated with the src family protein tyrosine kinase p56lck (8, 9). Monocytes/macrophages express several src family protein tyrosine kinases, including fgr, fyn, hck, and lyn (34). LPS- and IFN-{gamma}-stimulated macrophages up-regulate hck and lyn (26). In addition, hck is involved in TNF production by macrophages (35). Genistein, a broad spectrum tyrosine kinase inhibitor, inhibited OX8 (anti-CD8{alpha})- and 341 (anti-CD8ß)-stimulated NO production and iNOS up-regulation (Figs. 3Go and 4Go). To determine whether the kinase(s) involved in CD8-mediated stimulation belongs to the src family, the inhibitor PP1 was used. PP1 dose dependently inhibited OX8- and 341-mediated NO production and iNOS up-regulation (Figs. 5Go and 6Go). These results suggest that src family kinases are involved in CD8-mediated stimulation of alveolar macrophages, similar to T lymphocyte CD8{alpha}-mediated stimulation. However, the inhibition of CD8ß-stimulated macrophage function by PP1 further suggests differences between macrophage and T lymphocyte CD8ß. Macrophage CD8ß may directly associate with a src family kinase to stimulate mediator release independent of CD8{alpha}. Alternatively, macrophage CD8ß may also function through CD8{alpha} to induce mediator release. T lymphocyte CD8ß can associate with the src family kinase p56lck, but this may increase p56lck association with CD8{alpha} (36), thus enhancing the effector function of CD8{alpha}.

Further studies on the signaling pathways of CD8 concentrated on PKC. Our studies using polymyxin B and Ro 31-8220 (Figs. 3Go and 4Go) demonstrated that CD8{alpha}- and CD8ß-induced NO stimulation and iNOS up-regulation are PKC dependent. These results correlate with those of Paul et al. (37) and Eason and Martin (15), who demonstrated that LPS- and IFN-{gamma}-induced macrophage iNOS up-regulation is PKC dependent.

From these results, we have put forth two working models for CD8 stimulation (Fig. 7Go). In model one, CD8 may stimulate macrophage function directly through src family kinase and PKC-dependent pathway(s) (Fig. 7GoA). In model two, CD8 acts as a coreceptor for the Fc{gamma}R(s) expressed by macrophages (Fig. 7GoB). Macrophages express the high affinity receptor Fc{gamma}RI (38), and studies have shown that stimulating Fc{gamma}Rs can induce the expression of iNOS in rat peritoneal macrophages (39). In addition, Fc{gamma}RI receptors have been shown to associate with the src family kinases hck and lyn (40). Therefore, our results may be explained by CD8 acting as a coreceptor in conjunction with Fc{gamma} (Fig. 7GoB).



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  		FIGURE 7. Working models for CD8-mediated stimulation of NO and up-regulation of iNOS. A, CD8 directly stimulates iNOS and NO production in a protein tyrosine kinase (src family) and PKC-dependent pathway(s). B, CD8 acts as a coreceptor, signaling macrophage function in a protein tyrosine kinase (src family) and PKC-dependent pathway(s), in conjunction with Fc{gamma}.

 
Experiments on NO stimulation using an IgG anti-IgG model, used to examine the role of Fc{gamma}Rs, failed to initiate NO stimulation in alveolar macrophages (data not shown). In addition, studies have shown that PI3 kinase is involved in Fc{gamma}R-mediated signaling (41). Because we have evidence that PI3 kinase does not play a role in CD8-mediated NO stimulation or iNOS up-regulation, we hypothesize that CD8 functions independently of the Fc{gamma}R. Studies using F(ab')2 OX8 (anti-CD8{alpha}) will be used to test both these hypotheses on CD8 stimulation and will further elucidate which of these models best represents CD8-mediated signaling in macrophages.

Independent of whether CD8 activates macrophages directly or as a coreceptor, we have demonstrated that CD8 plays an important role in stimulating macrophage function. L. major-infected alveolar macrophages were stimulated to kill this parasite, which was NO dependent, when cross-linked with anti-CD8{alpha} (OX8) (Table IGo). Moreover, anti-CD8{alpha}-stimulated killing of Leishmania was not significantly different from IFN-{gamma} (200 U/ml)-mediated killing.

Our studies using L. major infection showed differences in OX8- and 341-stimulated NO release (Table IGo), compared with noninfected macrophages (Fig. 1Go), with OX8 stimulating more and 341 stimulating less NO/cell. One possible explanation for the differences in NO production could be that expression of CD8{alpha} and CD8ß is altered on infected macrophages, thereby changing the density of these surface molecules. Previous studies examining the expression of CD8 on uninfected macrophages (using an alveolar macrophage-rich population, 89 ± 1% OX41) showed that 63 ± 5% stained positive for OX8, and 52 ± 3% stained positive for 341 (6). In an attempt to determine whether parasitic infection and associated pulmonary inflammation could modify the expression of CD8 on alveolar macrophages, we infected rats with the nematode Nippostrongylus brasiliensis. No significant difference in the expression of CD8{alpha} or CD8ß was found (data not shown). However, as Leishmania infects macrophages, we cannot rule out the possibility that this parasite may alter CD8{alpha} and/or CD8ß expression.

These results stress the importance of CD8 expression on macrophages. Macrophage CD8 may interact with MHC I (ligand for T lymphocyte CD8) to form a more stable interaction between the APC and T lymphocytes and enhance macrophage adhesion. In addition, CD8 may also allow macrophages to modulate the immune response by inducing apoptosis in a subset of T lymphocytes. Sambhara and Miller (42) demonstrated that precursors to cytotoxic and Th lymphocytes can be deleted, by apoptosis, when signaled through their TCR and MHC I molecule. To perform efficient deletion of Th cells, Sambhara and Miller (42) postulated the need for a cell to express both MHC II and CD8, which macrophages have been shown to express. Alternatively, because macrophage and T lymphocyte CD8 differs within the ligand binding domain (6), macrophage CD8 may interact with a novel ligand(s). This would suggest a hitherto unknown role for macrophage CD8. Recent work has demonstrated a non-MHC I ligand for T lymphocyte CD8 (43), supporting the hypothesis for novel CD8 ligands.

For the first time, an effector function for both the {alpha}- and ß-chains of macrophage CD8 is demonstrated. Macrophage CD8 can regulate mediator release and stimulate macrophage host defense. In addition, the ß-chain of macrophage CD8 can also regulate effector function, further supporting our previous work (6) demonstrating differences between macrophage and T lymphocyte CD8. Further studies on the signaling mechanisms and ligand(s) for macrophage CD8 will lead to a greater understanding of macrophage-regulatory mechanisms and the role of CD8 in modulating immune responses.


   Acknowledgments
 
Anti-murine inducible nitric oxide synthase and peptide NO17 were provided by Dr. J. Weidner and Merck Research Laboratories. Ro 31-8220 was a kind gift from ROCHE.


   Footnotes
 
1 This work was supported by Alberta Lung Association and Medical Research Council of Canada. N.H. was funded by studentships from University of Alberta and Alberta Lung Association. Back

2 Address correspondence and reprint requests to Dr. A. Dean Befus, Pulmonary Research Group, The University of Alberta, Room 574 HMRC, Edmonton, Alberta, T6G 2S2, Canada. Back

3 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible nitric oxide synthase; NOS, nitric oxide synthase; PI3, phosphatidylinositol 3-kinase; PKC, protein kinase C. Back

Received for publication November 19, 1997. Accepted for publication February 9, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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