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Originally published online as doi:10.1189/jlb.1006640 on February 16, 2007

Published online before print February 16, 2007
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(Journal of Leukocyte Biology. 2007;81:1205-1212.)
© 2007 by Society for Leukocyte Biology

Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion

Therese A. Dietlin*, Florence M. Hofman*, Brett T. Lund*, Wendy Gilmore*, Stephen A. Stohlman{dagger} and Roel C. van der Veen*,1

* Departments of Neurology and Pathology, University of Southern California Keck School of Medicine, Los Angeles, California, USA; and
{dagger} Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA

1 Correspondence: Department of Neurology, University of Southern California Keck School of Medicine, MCA 245, 1333 San Pablo Street, Los Angeles, CA 90033, USA. E-mail: vanderve{at}usc.edu

ABSTRACT

Similar to the regulation of vasodilation, the balance between NO and superoxide (O2–) regulates expansion of activated T cells in mice. Reduction of suppressive NO levels by O2– is essential for T cell expansion and development of autoimmunity. In mice primed with heat-killed Mycobacterium, a splenocyte population positive for Gr-1 (Ly-6G/C) is the exclusive source of both immunoregulatory free radicals. Distinct Gr-1+ cell subpopulations were separated according to Ly-6G expression. In culture with activated T cells, predominantly monocytic Ly-6G– Gr-1+ cells produced T cell-inhibitory NO but no O2–. However, mostly granulocytic Ly-6G+ cells produced O2– simultaneously but had no measurable effect on proliferation. Recombination of the two purified Gr-1+ subpopulations restored controlled regulation of T cell proliferation through NO and O2– interaction. Coculture of p47phox–/– and inducible NO synthase–/– Gr-1+ cells confirmed this intercellular interaction. These data suggest that bacterial products induce development of distinct Gr-1+ myeloid lineages, which upon stimulation by activated T cells, interact via their respective free radical products to modulate T cell expansion.

Key Words: nitric oxide • superoxide • myeloid suppressor cells • granulocytes • monocytes

INTRODUCTION

The enzymatically produced free radical superoxide (O2–) and NO are considered host defense effector molecules. In addition, NO plays a major immunosuppressive role, mediating several traditional immunosuppressive strategies [1 2 3 4 ]. Mice deficient in inducible NO synthase (iNOS) develop exacerbated experimental immunopathologies, including myasthenia gravis [5 ], arthritis [6 ], myocarditis [7 ], and experimental autoimmune encephalomyelitis (EAE) [8 9 10 ], as well as increased T cell responses during parasitic infections [11 ]. NO induction during T cell activation inhibits subsequent T cell expansion and has been implicated as the mechanism of NO-mediated immunosuppression in vivo [2 , 5 , 12 13 14 15 ].

NO and O2– mutually react with high affinity to form peroxynitrite, which regulates NO bioavailability during such NO-dependent processes as vasodilation [16 17 18 ], inhibition of endothelial cell growth [19 ], and inhibition of neuronal respiration during inflammation [20 ]. Although in recent years, this process became widely studied in vascular biology, it has received little notice in immunology, despite a large body of work describing immunosuppression by NO. However, the interaction of NO and O2–, induced simultaneously during splenic T cell activation, plays a role in immune regulation as well. For example, NO interaction with O2– modifies leukocyte recruitment [21 ] and limits NO-dependent inhibition of T cell proliferation [22 ]. It is notable that O2– production by NADPH oxidase during the sensitization phase, not during inflammation, is essential for the development of EAE [22 23 24 ]. This was demonstrated in adoptive transfer, where inhibition of NO production during the sensitization phase is sufficient to revert the EAE-resistant phenotype of NADPH oxidase-deficient mice into the highly susceptible wild-type phenotype [23 ]. Thus, O2– production by NADPH oxidase is not required for inflammation and severe EAE development, but is required for diminishing NO levels and its inhibition of T cell expansion during the sensitization stage of EAE induction, similar to its role in vasoconstriction. This exemplifies the significance of the NO–O2– interaction in the management of immune response induction.

Alternatively, O2– is converted to hydrogen peroxide by O2– dismutase (SOD). As production of O2– by NADPH oxidase is restricted to the extracellular compartment, exogenous SOD enhances its dismutation, resulting in elevated NO levels and enhanced suppression of T cell proliferation [23 ]. In our culture system, the major function of exogenous SOD may be to enhance NO bioavailability, rather than act as an antioxidant.

The significance of the interaction between NO and O2– to immune regulation in vivo indicates that identification of their source is essential to the understanding of this regulatory mechanism. A population of Gr-1+ CD11b+ myeloid cells, identified as myeloid suppressor cells (MSC), is an established, major source of NO, which develops following stressors as varied as tumors, parasites, or radiation in mice [25 26 27 28 29 30 31 ]. Priming of mice with CFA, containing mycobacterial products, induces development of specialized Gr-1+ cell populations, which can subsequently be stimulated by activated T cells to produce O2– and NO. Gr-1+ splenocyte populations derived from control primed (adjuvant without mycobacterial products) or naïve mice produce neither O2– nor NO [29 ], indicating that mycobacterial products are required in their development. These Gr-1+CD11b+ cells, whose products determine to what extent T cell expansion is suppressed, were tentatively termed Greg as a functional distinction from nonregulatory Gr-1 cells. Conceivably, Greg are involved in the regulation of immune response induction in vivo, as their production of NO and its antagonist O2– has important opposing consequences for EAE induction.

It is unknown whether a single subpopulation within Gr-1+ cells produces immunoregulatory free radicals. In the present report, primed Gr-1 (Ly-6G/C)+ cells were separated into two phenotypically distinct subpopulations, distinguished primarily by differential expression of Ly-6G, a subspecificity of Gr-1. Functionally, Ly-6G– Gr-1+ cells produced NO during T cell activation, strongly inhibited proliferation, and expressed a predominantly monocytic phenotype. By contrast, mostly granulocytic Ly-6G+ cells exhibited no T cell inhibitory capability, correlating with their low NO production; however, during T cell activation, this population produced O2–. Reconstitution of the two Gr-1+ fractions restored regulation of T cell proliferation through NO and O2– interactions, which was confirmed by cocultures of iNOS–/– and p47phox–/– Gr-1+ cells. These data suggest that distinct Gr-1+ myeloid lineages interact via their respective free-radical products, managing T cell expansion.

MATERIALS AND METHODS

Mice and reagents
C57BL/6 mice were obtained from Harlan (Indianapolis, IN, USA). DO11.10 OVA323–339 (OVA323) TCR transgenic mice were originally obtained from Dr. Ann O’Garra (DNAX Corp., Palo Alto, CA, USA) and maintained locally. Nos2<tm1Lau>/J (iNOS–/–) mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). NADPH oxidase-deficient (p47phox–/–) mice were kindly donated by Dr. Steven M. Holland (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA). The mutant strains were maintained in local facilities. The Institutional Animal Care and Use Committee approved all experiments.

Peptide OVA323 was synthesized by the Microchemical Core Facility at the University of Southern California Comprehensive Cancer Center (Los Angeles, CA, USA). N-monomethyl-l-arginine (L-NMA) was purchased from Alexis (San Diego, CA, USA). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA).

Flow cytometry
Cell surface expression was analyzed by FACS on a Becton Dickinson (San Jose, CA, USA) fluorescence analyzer, as described previously [22 ]. Antibodies included anti-Ly-6G (1A8), anti-Ly-6G/C (Gr-1, RB6-8C5), anti-Ly-6C (AL-21) and anti-CD11b (M1/70), anti-NK1.1 (PK136), anti-stem cell antigen 1 (anti-Sca-1; D7), and anti-CD31 (390; BD PharMingen, San Diego, CA, USA) and anti-F4/80 (CI:A3-1; Caltag Laboratories, Burlingame, CA, USA).

Gr-1+ spleen cell separation
Regulatory cells were induced in C57BL/6 mice by s.c. immunization at two to three dorsal sites with 0.2 ml IFA and buffered saline emulsion, supplemented with 500 µg Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA). After 10–14 days, total Gr-1+ cells were purified from splenocytes with RB6-8C5 mAb-coupled magnetic beads (IMag, BD PharMingen), according to the manufacturer’s instructions. To magnetically purify only Ly-6G+ cells directly, total splenocytes were incubated with R-PE-labeled mAb 1A8 (4 µg/ml, BD PharMingen;) for 30 min at 4°C, followed by incubation with anti-PE-labeled magnetic beads, also for 30 min at 4°C, according to the manufacturer’s instructions (BD PharMingen). Positively selected populations were 95–98% pure, as determined by flow cytometry using FITC-labeled 1A8 mAb. To obtain Ly-6G– Gr-1+ cells, Ly-6G-depleted splenocytes were magnetically enriched for Gr-1+ cells as described above. The Ly-6G–Gr-1+ population contained 5–15% Ly-6Glo cells.

Functional analysis of regulatory Gr-1 cell activity in proliferation assays
Gr-1+ subsets (0.5x106 cells/ml) purified from CFA-primed donors’ spleens were cultured with spleen cells from naïve DO11.10 mice (0.75x106 cells/ml) in standard 96-well plates at 0.2 ml/well at 37°C. Upon activation with 1 µg/ml OVA323, naïve DO11.10 splenic T cells, which do not produce NO or O2–, have a dual function in mixed cultures with regulatory Gr-1 cells. First, OVA-activated T cells stimulate primed Gr-1 cells to produce NO and O2–, and second, the extent of their subsequent proliferation is an indicator of Greg activity. Most, if not all, inhibition by Gr-1 cells is mediated by NO, as naïve DO11.10 splenocyte proliferation in the absence of Gr-1 cells is similar as in the presence of Gr-1 cells and L-NMA [29 ]. This heterogeneous culture system did not induce significant MLR, as T cell proliferation was minimal in the absence of antigen (ref. [29 ]; also see Fig. 2 ). The advantage of this culture system is the restriction of antigen-specific T cell activation to the allogeneic DO11.10 splenocytes, which enabled us to restrict the analysis of Gr-1+ cell populations specifically to their regulatory activity, without confounding antigen presentation activity.


Figure 2
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  		Figure 2. Phenotypic and functional differentiation of Gr-1+ subsets, separated by Ly-6G expression. (A) Gr-1+ cells were purified magnetically from splenocytes derived from CFA-primed donor mice and stained with FITC-labeled, Ly-6G-specific mAb 1A8 (open area) or isotype control mAb (shaded area) and analyzed by flow cytometry. Representative data from one of three separate experiments. (B) Ly-6G+ cells were magnetically purified from CFA-primed spleen cells stained with PE-labeled 1A8 mAb (right panel). Subsequently, Gr-1 cells were magnetically enriched from the residual Ly-6G– fraction (left panel). The separated Gr-1+ fractions were analyzed for the presence of PE-labeled cells without additional labeling and depicted as histograms. (C–E) Separated Ly-6G– Gr-1+ cells (left panels) or Ly-6G+ cells (right panels) were cultured in the presence of naïve DO11.10 spleen cells and OVA323. Cross-hatched bars, OVA323-stimulated DO11.10 [transgenic (TG)] cells alone; open bars, mixed cultures without OVA323 (with added L-NMA in C only, to demonstrate the relatively small contribution of allogeneic responses to proliferation); solid bars, OVA323-activated, mixed cultures; hatched bars, OVA323-activated and L-NMA-treated, mixed cultures. (C) NO-mediated suppression of T cell proliferation. (D) NO production measured as nitrite accumulation. (E) O2–-producing cell numbers, analyzed by NBT-SPOT assay. Mean results ± SD from triplicate cultures representative of five separate experiments.

After 48 h, cultures were pulsed with tritiated thymidine and incubated for an additional 16 h, followed by analysis of tritium incorporation in individual wells. To analyze proliferation in the absence of NO production, L-NMA (0.15 mM) was added 20 h after culture onset. To analyze the effect of O2– on proliferation, SOD (150 U/ml) was added at 20 h. SOD had no inhibitory activity in the presence of L-NMA (ref. [23 ] and data not shown). To measure nitrite production, supernatants from identical cultures were obtained 48 h after culture onset and analyzed with a Griess reagent kit according to the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA).

Culture medium consisted of RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.05 µM 2-ME, penicillin (100 U/ml), streptomycin (100 U/ml), and gentamycin (10 µg/ml) all from Life Technologies (Gaithersburg, MD, USA) and 10 mM HEPES (Sigma Chemical Co.).

Quantification of O2–-producing cells [nitroblue tetrazolium-ELISPOT (NBT-SPOT)]
To analyze the number of O2–-producing cells, parallel cultures, under conditions identical to the proliferation assays described above, were incubated in 96-well Multiscreen-HA plates (Millipore, Watertown, MA, USA) in triplicate in the presence or absence of antigen, as described [23 ]. After 23 h, NBT (12.5 µg/ml, KPL, Gaithersburg, MD, USA) was added, and incubation continued for 1 h, followed by washing and drying of the wells. SOD (150 U/ml) was added simultaneously with NBT to confirm O2– as a reducing agent. Spots were quantified on a KS ELISPOT Assay System (Zeiss, Thornwood, NY, USA) with parameters set to include spots of 15–100 µm and varying from 60% to 100% circular shape. Results are presented as O2–-forming units per well, containing 105 Gr-1+ cells.

Hematological analysis
Cytospin preparations of Gr-1+ fractions were stained with H&E and analyzed by light microscopy. Mature granulocytes included band cells and PMN with segmented nuclei and neutrophilic granules; immature granulocytes included myelocytes and meta-myelocytes. Cells of the monocytic lineage were not differentiated further.

RESULTS

Relative quantities of Gr-1+ subsets in vivo
To examine the existence of Gr-1+ spleen cell subsets, distinguished by Ly-6G expression, we examined the distribution of Ly-6G and Gr-1 markers in spleens of naïve and CFA-primed mice by FACS analysis (Fig. 1 ). Distinct populations of Ly-6G+ and Ly-6G– Gr-1+ cells were observed in both groups. In naïve mice, Ly-6G+ and Ly-6G– Gr-1+ cells were present in low numbers, and the Ly-6G– Gr-1+ cells outnumbered their counterparts. In CFA-primed mice, both subsets comprised higher proportions of the total spleen cell population than in naïve mice and the strongest increase in Ly-6G+ cells. However, changes in fluorescence intensity for Ly-6G and Gr-1 induced by priming were slight, and mostly occurred in the monocytic population, compared with the corresponding populations derived from naïve donors. Naïve Gr-1+ cells do not exhibit any NO-dependent inhibition of T cell proliferation [29 ], suggesting the absence of a correlation between Gr-1 expression intensity and the acquired ability to produce O2– or NO in response to activated T cells.


Figure 1
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  		Figure 1. Development of splenic Ly-6G+ and Ly-6G– Gr-1+ subsets. Total spleen cell suspensions from naïve mice (left panel) or CFA-primed mice obtained after 10 days (right panel) were stained with FITC-labeled, anti-Ly-6G and allophycocyanin (APC)-labeled, anti-Gr-1 mAb and analyzed by flow cytometry. Within each panel, the upper two quadrants depict the monocytic Ly-6G– (left quadrant) and granulocytic Ly-6G+ (right quadrant) Gr-1+ subsets, respectively. Numbers in individual quadrants indicate the percentage of the total spleen cell population. Representative data of four experiments.

Distinct Gr-1+ subpopulations in spleens of CFA-primed mice
Upon purification, Gr-1+ spleen cells from primed mice contained distinct Ly-6Ghi and Ly-6Glo/– subpopulations as well (Fig. 2A ). Magnetically purified Ly-6G+ cells exhibited the Ly-6Ghi phenotype of total Gr-1 cells (Fig. 2B , right panel); however, this population exhibited no ability to inhibit T cell proliferation or produce NO and contained essentially all O2–-producing cells found within the unfractionated Gr-1+ population cultured with activated T cells (Fig. 2C 2D 2E , right column of panels). Addition of SOD did not alter proliferation (results not shown), confirming the absence of NO production by Ly-6G+ cells, rather than inactivation of NO via elevated O2– production by the same population. As Gr-1+ cells are the primary source of O2– and NO [29 ], we examined the Ly-6G– Gr-1+ population for production of NO. Gr-1+ cells purified from Ly-6G-depleted splenocytes exhibited a Ly-6Glo/– phenotype (Fig. 2B , left panel). This population strongly inhibited T cell proliferation in the absence of a NOS inhibitor and produced elevated levels of NO while containing few O2–-producing cells (Fig. 2 , left panels). Thus, the complex Gr-1+ splenocyte population can be separated by differential Ly-6G expression into functionally distinct Greg subpopulations.

Distinct myeloid lineage of Ly-6G+ and Ly-6G– Gr-1+ cells
The striking, functional correlation with Ly-6G expression suggested distinct phenotypic differences as well. Scatter profiles indicated that the two Gr-1+ subsets are morphologically distinct. The Ly-6G+ population was predominantly composed of granulocytes, and the Ly-6G– Gr-1+ population exhibited scatter characteristics of monocytes, partially overlapping with lymphocytes (Fig. 3A ). Consistent with derivation from the monocyte lineage, Ly-6G– Gr-1+ cells expressed more F4/80 than Ly-6G+ cells (Fig. 3B) . Other cell surface markers, including CD11b and Ly-6C, were expressed at high levels on both populations; NK1.1 expression was undetectable on either, and the immature markers CD31 and Sca-1 were expressed weakly on a small number of cells from both subsets (results not shown).


Figure 3
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  		Figure 3. Phenotypic characterization of separated Greg subsets. Magnetically purified Ly-6G– Gr-1+ (left panels) and Ly-6G+ (right panels) splenocyte fractions derived from CFA-primed mice (see Materials and Methods) were analyzed by flow cytometry. (A) Scatter profiles. (B) F4/80 expression after staining with APC-labeled mAb CI:A3-1. Numbers within histograms indicate percentage of F4/80+ cells. (C) Cytospin preparations from both fractions were analyzed by light microscopy (200x original magnification), and differential counts were made for monocytic (solid bars) and granulocytic cells (open bars; mean percentage of total cells in each subset±SD from three separate preparations).

Analysis of the morphological phenotype of the Ly-6G+ and Ly-6G– Gr-1+ populations was consistent with the flow-cytometric analysis. The Ly-6G+ subpopulation predominantly exhibited the phenotype of late maturing granulocytic cells, including many band cells and some PMN (results not shown). By contrast, the Ly-6G– Gr-1+ population contained predominantly monocytic cells in addition to a smaller fraction of cells with the morphology of immature granulocytes, including myelocytes (Fig. 3C) . Thus, Greg constitutes a system, which is comprised of two distinct myeloid lineages with opposing functions in T cell regulation: NO-producing Ly-6G– Gr-1+ monocytic cells and O2–-producing Ly-6G+Gr-1+ granulocytic cells.

Ly-6G+ and Ly-6G– Gr-1+ cells interact to regulate T cell expansion
To confirm that the two distinct Greg populations interact in the regulation of T cell expansion, a fixed concentration of NO-producing Ly-6G– Gr-1+ cells was cultured in the presence of increasing numbers of O2–-producing Ly-6G+ cells. Ly-6G– Gr-1+ cells alone inhibited antigen-specific T cell proliferation maximally in a NO-dependent manner (Fig. 4 ). Addition of Ly-6G+ cells at a ratio of 2:5 Ly-6G– Gr-1+ cells reduced NO-mediated inhibition, and the extent of reduction increased when the ratio was increased to 1:1. As reversal of NO activity was still incomplete with equal O2–-producing cells, we may conclude that NO-producing cells are functionally dominant. Addition of SOD restored most of the NO-mediated suppression, confirming that extracellular O2– produced by Ly-6G+ cells was essential to T cell proliferation. Thus, NO-dependent inhibition of T cell expansion is determined by the balance of NO and O2– produced by two morphologically and functionally distinct Gr-1+ populations.


Figure 4
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  		Figure 4. O2–-producing Greg reduce inhibitory activity from NO-producing Greg. Mixtures of purified Ly-6G+ and Ly-6G– Gr-1+ cells were reconstituted by adding Ly-6G+ cells at the indicated concentrations to mixed cultures of Ly-6G– Gr-1+ cells (0.5x106 cells/ml) and OVA323-activated, naïve DO11.10 splenocytes as stimulators and targets of Greg activity. NO-dependent inhibition of T cell expansion is indicated by the difference in proliferation in the presence or absence of L-NMA or SOD (see Materials and Methods). Open bars, No antigen; solid bars, OVA323-activated; hatched bars, OVA323-activated and L-NMA-treated; cross-hatched bars, OVA323-activated and SOD-treated. SOD had no inhibitory activity in the presence of L-NMA (not shown), indicating the role of NO in the enhanced inhibition by SOD. *, P = 0.004; **, P < 0.001, both compared with control cultures without Ly-6G+ cells. **, Compared with *, P = 0.003 (Student’s t test). Mean results of triplicate cultures representative of three separate experiments.

To confirm that iNOS and NADPH oxidase were the sources of NO and O2– produced by Greg, we isolated Gr-1+ splenocytes from CFA-primed iNOS–/– and p47phox–/– mice and cultured these with naïve DO11.10 splenocytes. In the presence of OVA, Gr-1+ splenocytes from iNOS–/– mice produced only O2– and supported maximum proliferation, and those from p47phox–/– mice produced no O2– but inhibited proliferation in a NO-dependent manner as expected (Fig. 5A and 5B ), confirming iNOS and NADPH oxidase as the major sources of regulatory NO and O2–, respectively. When varied numbers of Gr-1+ cells from O2–-producing iNOS–/– mice were cultured with a fixed number of NO-producing p47phox–/– Gr-1+ cells in the presence of activated, naïve DO11.10 splenocytes, the strong, NO-mediated suppression by p47phox–/– Greg alone was reduced gradually as O2–-producing iNOS–/– Gr-1+ cell numbers increased (Fig. 5C) , confirming that interacting NO and O2– arise from different cells.


Figure 5
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  		Figure 5. Gr-1+ cells from iNOS–/– and p47phox–/– mice as T cell regulators. Gr-1+ splenocytes were isolated from CFA-primed iNOS–/– and p47phox–/– mice and incubated separately (A and B) or together (C). All cultures contained OVA323-activated, naïve DO11.10 splenocytes (see Materials and Methods). (A) NO-mediated suppression of T cell proliferation, demonstrated by the difference in proliferation with or without L-NMA. (B) O2–-forming units, analyzed by NBT-SPOT assay. (C) Interaction between NO and O2–-producing cells in T cell regulation. Indicated numbers of iNOS–/– Gr-1+ cells were cocultured with a fixed number of p47phox–/– Gr-1+ cells (0.6x106/ml) and OVA323-activated, naïve DO11.10 splenocytes. The degree of proliferation is represented as percentage of maximal proliferation (i.e., in the presence of L-NMA: 36±1, 37±1, 39±3, and 31±2x103 cpm at 0.0, 0.2, 0.4, and 0.6x106 iNOS–/– Gr-1+ cells/ml, respectively). For a full explanation of bar patterns, see the legend to Figure 4 . Proliferation in the absence of antigen was insignificant and is not shown. Mean results of triplicate cultures ± SD representative of three separate experiments.

DISCUSSION

iNOS induction by activated T cells and subsequent suppression of T cell expansion are thought to be mechanisms responsible for immunosuppression by NO in vivo [2 , 5 , 12 13 14 15 ]. Simultaneously with NO production, O2– production by NADPH oxidase increased as well. Reaction of O2– with NO relieves suppression of T cell expansion, thereby permitting proliferation to proceed in the presence of NO-producing cells [22 , 23 ]. Reduction of O2– levels by SOD or in p47phox–/– mice results in enhanced suppression [22 ], indicating that peroxynitrite, the product of this reaction, does not impair proliferation in this system, as in that case, NO and O2– would promote inhibition. Our data and those of others [16 17 18 19 20 21 22 23 ] suggest that the major consequence of NO–O2– interaction in general may be a reduction in NO bioavailability. However, its relevance for immunoregulation has not been widely recognized, despite wide recognition of suppression by NO.

A related form of immunosuppression is arginine depletion by MSC-derived arginase [32 33 34 ], which reduces NO production. Addition of exogenous arginine reverses suppression by arginase; however, during NO-mediated immunosuppression by Greg, arginine supplementation increases NO production and enhances suppression, as can be expected with increased substrate (data not shown). In this intercellular process, O2– is thought to regulate bioavailability of NO after its production and diffusion into the extracellular compartment, where they interact. The existence of two separate processes regulating NO levels testifies to the significance of this immunosuppressor.

Gr-1+ MSC, which develop upon exposure to various types of environmental stressors, were identified previously as the source of immunosuppressive NO [25 26 27 28 29 30 31 ], but this Gr-1+ population includes O2–-producing cells as well [27 28 29 ]. We tentatively designated these Gr-1+ cells Greg [29 ], as concerted action through their respective products, which are induced by activated (but not resting) T cells, determines the degree of NO-dependent inhibition of T cell expansion, probably during immune response induction.

Using cocultures with naïve DO11.10 splenocytes as stimulators and indicators of Greg activity in purified Gr-1 populations from primed mice, we found that predominantly monocyte-like Ly-6G–Gr-1+ cells produce NO and suppress T cell proliferation and are probably equivalent to MSC [35 ]. Mostly granulocytic Ly-6G+ cells, conversely, produce O2– and do not suppress T cell proliferation. Instead, they behave as counter-suppressors, as their O2– production reduces NO bioavailability. Through their mutually exclusive and opposing activities, the two subsets act in concert to regulate T cell expansion (Fig. 6 ). Our current definition of Greg is as follows: an innate, immunoregulatory system consisting of Gr-1+ cells from two separate myeloid lineages (monocytic suppressor and granulocytic counter-suppressor), which develops upon exposure to mycobacterial products and is stimulated by activated T cells to produce NO or O2–, whose interaction determines the degree of proliferation of the activated T cells. Its function is independent of histocompatability, although its stimulation is indirectly antigen-dependent. Interaction of NO and O2– produced by separate populations was confirmed by mixed cultures consisting of Gr-1+ splenocytes from iNOS–/– and p47phox–/– mice, each capable of producing only one of the two free-radical species involved, thus confirming their respective enzymatic sources.


Figure 6
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  		Figure 6. The Greg concept. Antigen-activated T cells induce simultaneous NO and O2– production by monocytic and granulocytic Greg, respectively, whose development is determined by exposure to mycobacterial products. O2– reacts with NO to form peroxynitrite (ONOO–), reducing NO bioavailability. This interaction determines the extent of activated T cell expansion during immune response induction. Closed arrowheads indicate sequential processes; open arrowheads indicate stimulating pathways.

NO production by monocytes and O2– production by granulocytes are well established. Both lineages are represented in Gr-1+ myeloid cell populations in tumor-bearing mice, where both free-radical products may be immunosuppressive [27 ]. Their integration into an interactive myeloid system, which is stimulated by activated T cells and regulates T cell expansion, as proposed here, is novel. Stimulation of each subset by activated T cells is independent of the complementary subset; it is only after stimulation that proximity of the subsets is thought to be required to allow interaction between their products. Regulatory interaction of NO and O2– derived from different cells is an established process in vascular biology [17 , 18 , 36 ]. Although O2– derived from NADPH oxidase is reactive and restricted to the extracellular space, NO is comparatively stable and uncharged, allowing it to diffuse across cell membranes and over relatively long distances. Consequently, we expect the interaction to occur proximal to the cell membrane of the O2–-producing cells, where NO is captured and converted to peroxynitrite prior to reaching the activated T cells and preventing their expansion. In support of this hypothesis, addition of exogenous SOD protein, which does not penetrate the cell membrane, blocks T cell-induced O2– activity and rescues NO activity [29 ].

In culture with activated T cells, Gr-1+ splenocytes from naïve mice produce neither NO nor O2–, even at elevated cell numbers [29 ], demonstrating that bacterially induced Greg are not merely quantitatively but also qualitatively different from pre-existing Gr-1 splenocytes in naïve mice. Greg may differentiate from precursor cells, which have been reported in spleen and bone marrow [37 ]. An immature phenotype for inhibitory Gr-1+ cells was reported [27 , 30 , 31 ]. Gr-1 is absent on undifferentiated myeloid precursors and becomes permanently expressed during granulocytopoiesis, and it is expressed only transiently during monocyte/macrophage development [38 ].

A bacterial requirement for the development of NO-mediated inhibition of EAE by Gr-1 cells was described previously [39 ]. Greg development may be a general consequence of immunization, which includes CFA as adjuvant. In addition, similar consequences may result from infections or other stressors. Specific microbial or environmental products conceivably influence Greg development to give rise to Greg patterns and immunoregulation characteristics particular to the specific stressor. For instance, burn injury, which results in NO-mediated immune suppression [40 ], induces a myeloid commitment shift toward monocytopoiesis [41 ], consistent with dominance of NO-producing Greg over O2–-producing, granulocytic Greg. Conversely, factors that promote granulocyte maturation specifically, such as IL-17 via G-CSF [42 ], may preferentially promote development of the O2–-producing (i.e., immunostimulatory) Greg subset required for EAE. It is interesting that IL-17 is required for autoimmune inflammation [43 ].

Anti-Gr-1 mAb treatment reduces EAE severity only when applied during the effector phase, suggesting a requirement for Gr-1+ PMN during CNS inflammation [44 ]. This does not contradict our results, as Greg activity appears to occur earlier during the induction phase, and anti-Gr-1 treatment during the induction phase of EAE can be expected to deplete stimulatory and inhibitory Greg subsets, thereby exerting a limited net effect on the sensitization phase of EAE, and EAE development in NADPH oxidase-deficient mice, which lack O2–-producing cells, is suppressed as a result of unrestricted bioavailability of NO. Severe clinical and histological EAE develops in p47phox–/– mice by adoptive transfer of encephalitogenic donor cells, only when the donor splenocytes are activated in the presence of a NOS inhibitor [23 ].

These results illustrate the physiological relevance of the Greg system as the unique source of T cell-induced free radicals and suggest that a more important function of O2– may be the regulation of NO bioavailability during immune response induction, rather than a role during tissue inflammation. The sources of these physiologically important, immunoregulatory free radicals are identified here as unique components of a novel and complex innate system, whose development relies on environmental factors and which regulates EAE. This indirectly indicates the in vivo role of Greg. Matthys et al. [45 ] also suggested a dual response following immunization with CFA, resulting in the mycobacterium-dependent expansion of myeloid cells and production of IFN-{gamma}, in part overlapping with our postulate.

As the interactive Greg system has not been described before, many important questions remain. What is the site of T cell regulation by Greg in vivo? Do they develop in the bone marrow? It is interesting that NO-producing dendritic cell precursors develop in bone marrow cultures [35 ]. What is the relationship between subset maturation status and functionality? Greg may exist as a transient stage during myelopoiesis [35 ]. What are the factors that regulate the development of individual Greg subsets?

In conclusion, upon exposure to bacterial products, two Gr-1+ splenocyte populations representing different myeloid lineages develop T cell regulatory activity via exclusive production of interacting free radicals, induced by activated T cells. Differences in developmental mechanisms between the Greg lineages may offer future therapeutic strategies.

ACKNOWLEDGEMENTS

This work was supported in part by grant RG3476A12/1 from the National Multiple Sclerosis Society (R. C. V.). We are grateful to Drs. J. D. Crapo and S. M. Holland for helpful discussions.

Received October 18, 2006; revised January 16, 2007; accepted January 17, 2007.

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