Stress Cause T Cell Dysfunction After Traumatic Myeloid Suppressor Cells + /Gr-1 + Cd11b

The Journal of Immunology

CD11b؉/Gr-1؉ Myeloid Suppressor Cells Cause T Cell Dysfunction after Traumatic Stress1

Valeriya P. Makarenkova,* Vishal Bansal,* Benjamin M. Matta,* Lori Ann Perez,† and Juan B. Ochoa2*

T cell dysfunction that occurs after surgery or trauma is associated with a poor clinical outcome. We describe that myeloid suppressor cells expressing CD11b؉/Gr-1؉ markers invade the spleen after traumatic stress and suppress T cell function through the production of arginase 1. We created a consistent model of traumatic stress in C57BL/6 mice to perform this work. A signiﬁcant number of CD11b؉/Gr-1؉ cells expressing arginase 1 accumulated in T cell zones around the germinal centers of the white pulp of the spleen within6hoftrauma and lasted for at least 72 h. Increased arginase activity and arginase 1 expression, 3 along with increased [ H]arginine uptake, L-arginine depletion, and L-ornithine accumulation in the culture medium, were observed exclusively in CD11b؉/Gr-1؉ cells after traumatic stress. Flow cytometry revealed CD11b؉/Gr-1؉ as a heterogeneous myeloid suppressor cell also expressing low levels of MHC class I and II, CD80, CD86, CD31, and others. When compared with -controls, trauma-induced CD11b؉/Gr-1؉ cells signiﬁcantly inhibited CD3/CD28-mediated T cell proliferation, TCR ␨-chain ex pression, and IL-2 production. The suppressive effects by trauma CD11b؉/Gr-1؉ cells were overcome with the arginase antagonist N-hydroxy-nor-L-arginine or extrasupplementation of medium with L-arginine. Poor Ag-presenting capacity of control and trau- /ma-induced CD11b؉/Gr-1؉ cells was detected in allogeneic murine leukocyte reaction. This study demonstrates that CD11b؉ ,Gr-1؉ cells invade the spleen following traumatic stress and cause T cell dysfunction by an arginase-mediated mechanism probably that of arginine depletion. Understanding the mechanism of immune suppression by these cells has important clinical implications in the treatment of immune dysfunction after trauma or surgery. The Journal of Immunology, 2006, 176: 2085–2094.

atients who suffer severe trauma or have undergone major Expression of arginase 1 (ARG1),3 an enzyme that catabolizes surgery (from here on called traumatic stress) frequently arginine to ornithine and urea, is increased in peripheral mononu- P develop sepsis. Despite signiﬁcant progress, sepsis still oc- clear cells in humans and in splenic cells in mice after traumatic curs after traumatic stress, being an important contributing factor stress (9, 12). ARG1 expressed in myeloid cells in the immune in up to 14% of the in-hospital deaths of trauma patients (1). Im- tissues is associated with increased destruction of arginine (13, paired host defenses, frequently observed as T cell dysfunction, are 14). Thus, we hypothesized that arginine depletion by myeloid central to the development of infections after traumatic stress (2, cells expressing ARG1 could explain at least some elements of T 3). Despite its known importance, strategies aimed at restoring cell dysfunction after traumatic stress. immune function are limited, due in part to poor understanding of We report here that ARG1 expressed in immune tissues after its causes. trauma is observed exclusively in myeloid cells. Soon after trauma, T cell dysfunction after traumatic stress is characterized by de- an “invasion” of CD11bϩ/Gr-1ϩ cells is observed in splenic tis- creased T cell proliferation, production of cytokines (IL-2 and sues. These cells express very high levels of ARG1 and arginase IFN-␥), and a decreased expression of the TCR due to loss of the activity and exhibit increased arginine uptake (as measured by 3 ␨-chain peptide (4, 5). Withholding the amino acid arginine from [ H]arginine incorporation in vitro) as well as increased L-arginine the culture medium can partially reproduce these changes (6–8). depletion from the culture medium. In trauma, CD11bϩ/Gr-1ϩ co- We have described previously that arginine levels are dramatically localize with T cells around the germinal centers of the white pulp decreased after traumatic stress (9). Arginine levels recover only of the spleen. Trauma-induced CD11bϩ/Gr-1ϩ cells also express with its supplementation in the diet at supraphysiologic quantities MHC class I molecules but express low MHC class II, CD80, (10). Not surprisingly, the use of arginine is now shown to restore CD86, CD34, CD16/32, F4/80, and CD31. CD11bϩ/Gr-1ϩ/ T cell function after surgery and to decrease infection rates in these ARG1ϩ cells placed in the upper chamber of a Transwell and patients (11). cocultured in vitro with CD3/CD28-stimulated naive (nontrauma) T cells severely impair T cell proliferation, production of IL-2, and decrease TCR ␨-chain. These changes can be reversed through *Department of Surgery and †Department of Pathology, University of Pittsburgh pharmacologic blockade of ARG1 by N-hydroxy-nor-L-arginine Medical Center, Pittsburgh, PA 15213 (nor-NOHA) or by extrasupplementation of medium with 1.2 mM ϩ ϩ Received for publication November 4, 2004. Accepted for publication October L-arginine. CD11b /Gr-1 cells poorly stimulate proliferation of 30, 2005. naive allogeneic T cells. The costs of publication of this article were defrayed in part by the payment of page To our knowledge, this is the ﬁrst report demonstrating that charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ARG1-expressing myeloid cells can be a cause of T cell dysfunction after traumatic stress. Furthermore, our work is important in 1 This work was supported in part by National Institutes of Health Grant K08-646-03 (to J.B.O.) and National Institute of General Medical Sciences Grant R01-GM065914-01. 2 Address correspondence and reprint requests to Dr. Juan B. Ochoa, F1265 PUH-UPMC, 3 Abbreviations used in this paper: ARG1, arginase 1; nor-NOHA, N-hydroxy-nor- 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: [email protected] L-arginine; DC, dendritic cell.

that it demonstrates an otherwise easy and reproducible murine (25 ␮l) and incubation at 55°C for 20 min. Carbonate buffer (150 ␮l, 100 model of traumatic stress that will allow us to better understand mmol/L, pH 10) was then added along with 100 mmol/L L-arginine (50 ␮l) immune dysfunction in this disease process. to initiate the reaction, and incubated at 37°C. Arginase activity was stopped after 10 min by adding glacial acetic acid (750 ␮l). Ninhydrin solution (250 ␮l) (2.5 g of ninhydrin, 40 ml of 6M phosphoric acid, and 60 Materials and Methods ml of glacial acetic acid) was added, and the samples and standards were Mice boiled at 90–100°C for 1 h. Standards were created by using known amounts of L-ornithine from 8 to 250 nmol, and all reagents were added to Male C57BL/6 mice were obtained from Charles River Laboratories. Four standards as a control. Samples were cooled and colorimetric reaction mea- mice per cage were housed and maintained under a 12-h light/dark cycle at sured with a spectrophotometer at 515 nm (Spectramax 340; Molecular a temperature of 20–22°C in a pathogen-free facility. Food and water were Devices). Arginase assay was linear with time and is presented as nano- available ad libitum. The mice were allowed an acclimation period of 2 wk moles of ornithine per minute per milligram of protein. and used at 6–8 wk of age. Measurement of [3H]arginine uptake by CD11bϩ/Gr-1ϩ cells Mouse traumatic stress model CD11bϩ/Gr-1ϩ cells were isolated from splenocytes 24 h after laparotomy The experimental protocol was approved by the University of Pittsburgh and control also from mice that received anesthesia only. Cells were placed Institutional Animal Care and Use Committee and Division of Laboratory in a 96-well plate by 105 cells per 0.2 ml of RPMI 1640 medium without 3 Animal Research. Mice were randomized into two groups: a control group, L-arginine. Immediately, 5 ␮Ci of [ H]arginine (DuPont/NEN) was added receiving anesthesia alone, and an experimental group of mice undergoing to each well, and cells were incubated at 37°C. Cells were harvested at 10, traumatic stress. After administering the anesthetic (Nembutal, 50 mg/kg; 30, and 60 min, and isotope incorporation was measured using a 1450 Abbott Laboratories), a midline laparotomy incision was made. The intra- MicroBeta TRILUX liquid scintillation counter (Wallac) and quantified abdominal contents were teased for 15 s, taking care not to create injury to as cpm. the viscera. The incision was closed in two layers, and animals were maintained under a heat lamp until fully recovered from the anesthetic. Animals HPLC determination of L-arginine and L-ornithine in culture were sacrificed at 6, 12, 24, 48, or 72 h following laparotomy, and a sple- medium nectomy was performed for cell harvest. The L-arginine and L-ornithine concentration in tissue culture medium was Isolation of cells measured by HPLC with electron capture detection using an ESA-Cou- lArray Model 540 (ESA) with an 80 ϫ 3.2 column with 120A pore size. A single-cell suspension was prepared from the spleens of control and mice Briefly, supernatants were deproteinized in methanol. After centrifugation subjected to traumatic stress. Erythrocytes were depleted using RBC lysing at 6000 ϫ g for 10 min at 4°C, the supernatant was derivatized with 0.2 M buffer (Sigma-Aldrich), and splenocytes were washed in MACS buffer (1ϫ ϩ ϩ o-phthaldialdehyde containing 7 mM ␤-ME. Fifty microliters of the sample PBS supplemented with 2 mM EDTA and 0.5% BSA). CD11b , CD11c , were injected into the column. Standards of L-arginine and L-ornithine in NK, and T cells were isolated using corresponding MACS magnetic mi- methanol were run with each experiment. crobeads (Miltenyi Biotec). The purity of cells separation ranged between 89 and 95%. Flow cytometry analysis Protein extracts Harvested cells were washed in FACS medium (1ϫ PBS supplemented with 0.1% BSA and 0.1% NaN ) and stained with appropriately diluted Total cell protein extracts were prepared by lysing washed cell pellets in 10 3 Abs directly conjugated with FITC or PE according to the standard pro- ␮l of lysing buffer/106 cells (50 mM HEPES, 150 mM NaCl, 5 mM EDTA, cedure, and followed by fixation in 2% paraformaldehyde. Abs used for 1 mM NaOV , and 0.5% Triton X-100) containing 50 ␮g/ml aprotonin, 50 4 FACS staining were the following: FITC-labeled anti-mouse CD11b, ␮g/ml leupeptin (Roche), 100 ␮g/ml trypsin-chymotrypsin inhibitor, and 2 NK1.1, CD14, CD31, MHC class I, CD40, B220, CD4, CD80, CD36, mM PMSF (Sigma-Aldrich). Cell pellets were incubated on ice for 7 min CD34, CD131, CD13, CD68, F4/80, CD16/32, DEC-205, and PE-labeled with RBC lysing buffer and then centrifuged at 3,000 ϫ g (12,000 rpm) for GR1, CD86, CD11c, CD16, CD19, CD3, MHC class II, CD8␣ (BD Pharm- 15 min at 4°C. Total protein concentration was determined by the Bradford ingen). All staining procedures were conducted on ice. Fluorescence was method (15). Prepared protein lysates were aliquoted and stored at Ϫ80°C measured using a FACScan flow cytometer (BD Biosciences), and data until used for Western blot analysis or arginase activity assay. analysis was performed using CellQuest software (BD Biosciences). Cell Western blot analysis of ARG1 sorting was performed on a MoFlo cell sorter (DakoCytomation). Total protein from CD11bϩ, CD11cϩ, NK, and T cells were separated by Morphological analysis 12.5% SDS-acrylamide gel for 45 min at 200 V and electrophoretically ϩ One hundred microliters of control and traumatic stress-induced CD11b / transferred to a nitrocellulose membrane (Invitrogen Life Technologies). ϩ Gr-1 cell suspensions was loaded into a cytospin chamber and spun for 5 The protein extract from the mouse liver served as a positive control. The min at 500 rpm. Slides were air-dried at room temperature for 5 min and nitrocellulose membrane was blocked with 5% nonfat dry milk in TBST stained in a three-step procedure using the LeukoStat stain (Fisher (2.42 g of Tris,8gofNaCl, and 0.5 ml of Tween 20 in 1 liter of MQH O) 2 Scientific). at 4°C overnight. The membrane was incubated with chicken IgG1 anti- mouse ARG1 primary Ab (donated by Dr. S. Morris, Jr., University of T cell proliferation assay Pittsburgh, Pittsburgh, PA), diluted at 1/50,000 in 1% nonfat dried milk, 1% BSA in TBST, for1hatroom temperature (16). The membrane was A total of 1 ϫ 103–1 ϫ 106 CD11bϩ/Gr-1ϩ cells isolated from control or washed and the secondary peroxidase-conjugated rabbit anti-chicken IgG 24-h traumatic stress mice were cultured in the upper chamber of a Trans- Ab (Jackson ImmunoResearch Laboratories) was diluted 1/5,000 and ap- well (6-well plates), which has 0.4-␮m pores (Falcon/BD Biosciences) for plied for1hatroom temperature. As an internal control for total protein 24 h using RPMI 1640 medium containing 150 ␮M L-arginine (physio- concentration, the membrane was stripped and washed, and goat anti- logical levels). In parallel, 1 ϫ 106 normal splenic T cells were stimulated mouse ␤-actin Ab (Santa Cruz Biotechnology) was diluted 1/500 and ap- with 1 ␮g/ml anti-CD3 plus 1 ␮g/ml anti-CD28 (BD Pharmingen) in the plied for1hatroom temperature. After washing, the membrane was in- absence of L-arginine for 24 h. The stimulated T cells were then cultured cubated with goat anti-donkey IgG Ab (Santa Cruz Biotechnology), diluted in the bottom chamber of a Transwell system at 37°C in a humidified 5% 1/5000, and applied for 1 h at room temperature. For both ARG1 and CO2 atmosphere for 72 h. Nontreated T cells served as controls. Each well ␤-actin, immunoreactive protein was visualized using enhanced luminol was pulsed with 1 ␮Ci [3H]methyl-thymidine (DuPont/NEN) for the final reagent and oxidizing reagent (Pierce). Prestained Kaleidoscope molecular 18 h of incubation. Ten micromoles of nor-NOHA (Calbiochem) was mass marker (Bio-Rad) was used to determine the m.w. of immunoreactive added to block arginase, as well as medium containing 1.2 mM L-arginine bands. was used. Cells were harvested onto filtermates (Wallac), and isotope incorporation was measured by 1450 MicroBeta TRILUX liquid scintillation Arginase activity assay counter. Data are expressed as cpm Ϯ SEM. Arginase activity in protein lysates of splenocyte subsets was measured IL-2 production (ELISA) from the conversion of L-arginine to L-ornithine according to the technique described by Kornarska and Tomaszewski (17). Available arginase was To evaluate the effect of CD11bϩ/Gr-1ϩ cells on IL-2 production by Th1 ␮ activated by the addition of 10 mmol/L MnCl2 (25 l) to cell protein lysate cells, the level of IL-2 in cell-free supernatants was measured by ELISA The Journal of Immunology 2087 using the Mouse IL-2 Immunoassay Quantikine kit (R&D Systems). T cells Medium and reagents (106 T cells per 0.5 ml of medium per well) were placed in the bottom chamber of a Transwell (24-well plates) and stimulated with 1 ␮g/ml plate- RPMI 1640 medium (custom L-arginine free), L-arginine, L-glutamine, gen- bound anti-CD3 Ab and 1 ␮g/ml soluble anti-CD28 Ab (BD Pharmingen). tamicin, HEPES, FBS, nonessential amino acids, sodium pyruvate, and Control or traumatic stress-induced CD11bϩ/Gr-1ϩ cells (1 ϫ 106) were rabbit complement were purchased from Invitrogen Life Technologies; placed in 0.5 ml of medium in the upper chamber. Cell-free supernatants RBC lysing buffer, indomethacin, paraformaldehyde, and 2-ME were ob- were collected in 12 h and stored at Ϫ80°C. Ten micromoles of nor-NOHA tained from Sigma-Aldrich. (Calbiochem) was added to block arginase. Extrasupplementation of the Statistical analysis medium with 1.2 mM L-arginine also was used in selected experiments when appropriate. The sensitivity of the ELISA was 6 pg/ml. One-way ANOVA (SigmaStat software; Jandel) was performed to evaluate the significance of differences between the experimental groups. For a sin- Measurement of TCR ␨-chain gle comparison of two groups, Student’s t test was used after evaluation of normality. For all analyses, a probability level of 0.05 was considered 6 Naive T cells isolated from control mouse spleen (10 T cells per ml of significant. Data are presented as the mean Ϯ SEM. All experiments were medium per well) were placed in the bottom chamber of a Transwell (24- performed at least three times. well plates) and 106 control or traumatic stress-induced CD11bϩ/Gr-1ϩ cells were placed in 1 ml of medium in the upper chamber. Cells were Results cocultured for 24 h in RPMI 1640 medium containing 150 ␮MofL-arginine (physiologic level), 1 ␮g/ml plate-bound anti-CD3 Ab, and 1 ␮g/ml ARG1 expression after trauma is observed exclusively in ϩ soluble anti-CD28 Ab (BD Pharmingen) at 37°C in a humidified 5% CO2 CD11b cells atmosphere. Nontreated T cells cultured without CD11bϩ/Gr-1ϩ cells served as a control. Intracellular TCR ␨-chain expression in T cells was We have reported previously an up-regulation of arginase activity measured by flow cytometry using anti-mouse-CD3␨-PE Ab (Santa Cruz in murine splenic immune cells within 24 h of traumatic stress, Biotechnology). Rat-IgG2-PE Ab (BD Pharmingen) was used as isotype although the exact cell type expressing ARG1 was unknown (12, control. Ten micromoles of nor-NOHA were added to block arginase. Me- 18). To answer this question, we measured arginase activity in dium containing 1.2 mM L-arginine was used in additional experiments when appropriate. different cell subpopulations isolated through positive and/or negative selection using immunomagnetic beads for CD11bϩ, DCs ϩ Ϫ ϩ Ϫ ϩ Mixed lymphocyte reaction (CD11c /CD11b ), NK (DX5 /CD11b ), and T cells (CD3 ). ϩ ϩ Fig. 1 demonstrates that minimal baseline arginase activity was To study the Ag-presenting capacity of immature myeloid CD11b /Gr-1 detected in all splenocytes. However, we detected an increase in cells, MLR was performed using naive allogeneic T cell as target. Allo- ϩ geneic BALB/c T cells (H-2Kd) were isolated from the spleen by purifi- arginase activity in CD11b cells, which was 6-fold greater than cation on a nylon wool column. Control or traumatic stress-induced that of DCs and 40-fold higher than in NK and T cells ( p Ͻ 0.05). CD11bϩ/Gr-1ϩ cells were irradiated (3000 rad) and used as stimulator In fact, as can be seen in Fig. 1, arginase activity was 13-fold cells in the MLR assay. Dendritic cells (DCs) were isolated from control higher in splenic CD11bϩ cells obtained from animals after trau- spleens using CD11c MACS beads and served as a control. Stimulator cells ϩ Ϯ (102–105) were added to 2 ϫ 105 T cells per well in triplicate in 96-well matic stress, compared with control CD11b cells (412 49 round-bottom culture plates (Falcon) and incubated for 72 h in 200 ␮lof nmol/min/mg vs 17 Ϯ 12 nmol/min/mg; p Ͻ 0.05). Thus, trau- ␮ ϩ 150 M L-arginine medium at 37°C in a humidified 5% CO2 atmosphere. matic stress specifically induces arginase activity in CD11b cells. T cell proliferation was tested by [3H]thymidine incorporation. We further determined that increased arginase activity was due to the induction of ARG1 protein expression as determined by Immunohistochemical staining Western blot (Fig. 2). ARG1 expression was observed exclusively ϩ Immunohistochemistry was performed on mice sacrificed 6, 12, 24, or 48 h in splenic CD11b cells isolated from mice undergoing laparot- after laparotomy, and intact mice served as controls. Spleens were frozen omy. This is in accordance with previous results (Fig. 1). These in the presence of OCT (Sakura) and stored until used at Ϫ70°C. Cryostat results demonstrate that, between all tested splenocytes, only sections (4 ␮m) were used for immunohistochemical evaluation using rat ϩ CD11b cells express ARG1 after traumatic stress. This is in con- IgG2b,k anti-mouse Ly-6G (GR-1) mAb diluted 1/50 (BD Pharmingen) and ϩ goat polyclonal anti-mouse integrin ␣M (CD11b) diluted 1/50 (Santa Cruz trast with CD11b cells from control spleens, which do not ex- Biotechnology). Nonspecific binding was blocked with Super Block press significant amount of ARG1. Thus, traumatic stress induces (ScyTek Laboratories) for 8 min at room temperature, and sections were ARG1 in a specific subpopulation of myeloid cells. Arginase washed three times in 1% Tween 20/PBS buffer for 3 min. Sections were activity is proportional to ARG1 protein expression, supporting then incubated overnight in a humidified chamber at 4°C with primary rat anti-mouse Ly-6G (Gr-1) mAb (1.25 ␮g/ml). After three washes in Tween prior observations that arginase activity is transcriptionally con- 20/PBS buffer for 5 min, the sections were incubated for 30 min with the trolled (16). alkaline phosphatase-conjugated appropriated secondary Ab 1/200 or 3 ␮g/ml (Jackson ImmunoResearch Laboratories). The color reaction was developed for 8 min using alkaline phosphatase substrate kit III (Vector Laboratories). To stain the tissue for CD11b, sections were incubated overnight at 4°C with the goat polyclonal anti-mouse integrin ␣MAb(4␮g/ml) followed by quenching in 0.3% H2O2 solution for 10 min and blocking by avidin-biotin kit (Vector Laboratories). After washing in Tween 20/PBS buffer, the sections were incubated with the secondary biotinylated anti- goat Ab at dilution 1/200. Then, the sections were washed and incubated with avidin-biotin-HRP macromolecule complex (Vectastain, Elite ABC kit; Vector Laboratories), and CD11b-positive cells were visualized for peroxidase with the DAB substrate kit (Vector Laboratories). After the final wash in Tween 20/PBS buffer, all the sections were air-dried, dehy- drated, cleared, coverslipped, and studied under the microscope with bright field illumination. Control slides included an irrelevant isotype-matched Ab in place of the primary Ab: purified rat IgG isotype standard at a ϩ 1,k FIGURE 1. Arginase activity increases in splenic CD11b cells after 1/500 dilution (BD Pharmingen), goat IgG1,k isotype standard at a 1/500 ϩ dilution (BD Pharmingen), or PBS in place of the primary Ab to evaluate 24 h following surgical stress. Splenic CD11b , DCs, NK, and T cells were nonspecific staining. Control sections generally demonstrated negligible isolated using MACS beads 24 h after surgical stress. Control mice under- levels of endogenous background. Images were acquired from the double- went anesthesia alone. High levels of arginase activity (nanomoles per labeled sections using a confocal laser-scanning microscope (Olympus) at minute per milligram) were detected only after trauma. Trauma signifi- magnification ϫ20. cantly increases arginase activity only in CD11bϩ cells. (p Ͻ 0.0001). 2088 TRAUMA-INDUCED CD11bϩ/Gr-1ϩ CELLS SUPPRESS T CELL FUNCTIONS

FIGURE 2. Western blot for ARG1 following surgical stress. Equal amounts of protein from traumatic stress (A) and control (B) splenic CD11bϩ cells, DCs, NK, and T cells were blotted and detected using ARG1 Ab with liver protein lysate used as a positive control. ARG1 is a 35- to 38-kDa large protein. ␤-actin expression was used for semiquanta- tive control (C). Significant ARG1 expression was detected only in trauma ϩ CD11b cells compared with other cells. FIGURE 4. Increasing time after surgical stress increases arginase activity (A) and ARG1 expression (B) in splenic CD11bϩ/Gr-1ϩ cells. ϩ ϩ ϩ CD11b cells were separated from mouse spleen 6, 12, 24, 48, or 72 h after ARG1 expression is observed only in CD11b /GR-1 laparotomy. CD11bϩ/Gr-1ϩ cells obtained from mouse undergoing anes- splenocytes after traumatic stress thesia only were used as controls. Arginase activity was measured in cell Increased arginase activity has been described in immature my- lysates (nanomoles of ornithine produced per minute per milligram of pro- eloid cells expressing Gr-1 markers in murine models of cancer tein). A significant increase in arginase activity occurred with time after ,p Ͻ 0.05 ,ء) traumatic stress. Results are expressed as means Ϯ SEM (19). We performed the following experiments to determine ϩ ϩ ϩ compared with control CD11b /Gr-1 ). Western blot analysis confirmed whether CD11b cells expressing ARG1 after traumatic stress that the increase in arginase activity was due to increased ARG1 also were immature cells. Arginase activity was measured in expression. CD11bϩ/Gr-1ϩ and CD11bϩ/Gr-1Ϫ cell subpopulations sorted by flow cytometry from CD11bϩ splenocytes. Increased arginase activity (113 Ϯ 9 nmol/min/mg) was observed only in CD11bϩ/ ϩ cells at time intervals of 6, 12, 24, 48, and 72 h after laparotomy. Gr-1 cells harvested from animals subjected to traumatic stress, ϩ ϩ compared with control CD11bϩ/Gr-1ϩ cells or CD11bϩ/Gr-1Ϫ CD11b /Gr-1 cells isolated from mice undergoing anesthesia cells harvested from mice subjected to traumatic stress (Fig. 3). alone were used as controls. Fig. 4A demonstrates that arginase activity in trauma-induced These results reveal that, among all tested splenocyte subpopu- ϩ ϩ ϩ ϩ immature CD11b /Gr-1 cells increased linearly with time. An lations, only double-positive CD11b /Gr-1 cells exhibited an in- ϩ ϩ duction in ARG1 protein expression, and then only after traumatic early increase in CD11b /Gr-1 arginase activity occurred in the Ϯ stress. first 6 h, compared with control group (50 14 nmol/min/mg vs 15 Ϯ 7 nmol/min/mg). A significant increase in mean arginase ARG1 activity in CD11bϩ/Gr-1ϩ splenocytes increases linearly activity was maintained throughout 24 h after traumatic stress with time up to 72 h (396 Ϯ 55 nmol/min/mg) and increased to 732 Ϯ 87 nmol/min/mg B To examine the changes of arginase activity across time after trau- 72 h later. Fig. 4 demonstrates that there also was significant, matic stress, we examined arginase activity in CD11bϩ/Gr-1ϩ linear time-dependent increase in ARG1 protein expression in CD11bϩ/Gr-1ϩ cells induced by traumatic stress, as measured by Western blot. Therefore, we suggest that traumatic stress induces

FIGURE 3. Surgical stress increases arginase activity exclusively in splenic CD11bϩ/Gr-1ϩ cells. CD11bϩ cells were separated from control or 24 h posttraumatic stress mouse spleen and sorted by FACS to separate FIGURE 5. Traumatic stress-induced CD11bϩ/Gr-1ϩ cells character- ϩ ϩ ϩ Ϫ CD11b /Gr-1 and CD11b /Gr-1 cell subpopulations. Arginase activity ized by increased uptake of L-arginine. There is a signiﬁcant increase (p Ͻ (nanomoles per minute per milligram) was measured in cell lysates. High 0.05) in [3H]arginine incorporation in traumatic stress-induced CD11bϩ/ arginase activity was detected only in CD11bϩ/Gr-1ϩ cells following sur- Gr-1ϩ cells, compared with controls. [3H]arginine uptake was measured at gical stress (p Ͻ 0.0001, n ϭ 4 separate experiments). 10, 30, and 60 min. The Journal of Immunology 2089

trauma-induced CD11bϩ/Gr-1ϩ cells exhibited increased arginine incorporation, we measured [3H]arginine uptake in control and trauma CD11bϩ/Gr-1ϩ cells. The results show that CD11bϩ/ Gr-1ϩ cells harvested from mice 24 h after laparotomy had a significantly increased [3H]arginine uptake and incorporation, which was already present at 10 min but more clearly evident after 30 and 60 min of incubation (Fig. 5). For example, after 60 min in culture, ϩ the uptake of L-arginine by traumatic stress-induced CD11b / Gr-1ϩ cells was two times higher than in the same cells isolated from control mice (129,365 Ϯ 3,786 and 69,219 Ϯ 4,191 cpm, respectively; p Ͻ 0.05). This finding confirms that traumatic stress induces an increase in arginine uptake by CD11bϩ/Gr-1ϩ cells expressing ARG1.

ϩ ϩ CD11b /Gr-1 cells induced by traumatic stress deplete L- arginine from the culture medium by high arginase activity We then determined whether CD11bϩ/Gr-1ϩ myeloid cells were able to deplete arginine. To answer this question, we measured L-arginine concentration in culture medium. Control and traumatic stress-induced CD11bϩ/Gr-1ϩ cells were cultured for 48 h in 150 ␮M L-arginine medium, and concentrations of L-arginine and L- ornithine were measured in cell-free supernatants by HPLC analysis. As shown in Fig. 6A,2ϫ 106 CD11bϩ/Gr-1ϩ cells induced by traumatic stress deplete arginine from 150 ␮M down to 37 Ϯ 7 ␮M L-arginine, whereas the control cells exhibit negligible use of arginine. Importantly, the concentration of L-ornithine, a product of metabolism of L-arginine by ARG1, increased in culture medium of CD11bϩ/Gr-1ϩ trauma cells, corresponding to the de- ϩ ϩ FIGURE 6. Traumatic stress-induced CD11b /Gr-1 cells metabolize crease of L-arginine (Fig. 6B). Thus, our results are in agreement L-arginine (A)toL-ornithine (B) due to ARG1 in culture medium. L-Argi- with other ﬁndings that prove that traumatic stress-induced nine and L-ornithine concentration in tissue culture medium was measured ϩ ϩ CD11b /Gr-1 myeloid cells are able to deplete local L-arginine. by HPLC after 48 h of culture. Traumatic stress-induced CD11bϩ/Gr-1ϩ Ͻ cells depleted L-arginine, compared with control cells (p 0.05). At the Localization of CD11bϩ/Gr-1ϩ cells in mouse spleen detected same time, concentration of L-ornithine in culture medium of traumatic ϩ ϩ by immunohistochemistry stress-induced CD11b /Gr-1 cells was signiﬁcantly increased, compared with control cells. Several investigators, including us, have suggested that ARG1 regulates T cell function through arginine depletion in local micro- environments (6, 13, 14). For this hypothesis to be true, CD11bϩ/ an early and long-lasting arginase activity and ARG1 expression in Gr-1ϩ cells should localize at or near T cell-rich zones of the ϩ ϩ myeloid splenic CD11b /Gr-1 cells. spleen. To test this hypothesis, we performed serial immunohis-

ϩ ϩ tochemistry sections of splenic tissues after traumatic stress. L-Arginine uptake in trauma-induced CD11b /Gr-1 cells is ϩ ϩ ϩ Under resting conditions (controls), few Gr-1 cells were ob- increased, compared with control CD11b /Gr-1 cells served in splenic tissues and were mainly localized to the red pulp Induction of ARG1 with the use of IL-4 and IL-13 in mouse peri- (Fig. 7). A dramatic increase in Gr-1ϩ cells was seen within 6 and toneal macrophages is associated with increased [3H]arginine up- especially 12 h after traumatic stress and were tightly localized to take and incorporation. This also is associated with increased cat- the marginal zones and periarteriolar lymphatic sheaths of the ionic amino acid transporter expression. To determine whether spleen. By 24 and 48 h, the number of cells expressing Gr-1ϩ

FIGURE 7. Surgical stress causes an increase in GR-1ϩ cells in spleens. Frozen sections of spleens were stained with peroxidase-labeled anti-Gr-1 Ab (blue). Under resting conditions (control, anesthesia only), few Gr-1ϩ cells were observed mainly in the red pulp. A dramatic increase in Gr-1ϩ cells was seen within 6 and especially 12 h after traumatic stress and was circumscribed within the marginal zones and periarteriolar lymphatic sheaths of the spleen. By 24 and 48 h, the number of cells expressing Gr-1ϩ markers appeared to slowly decrease and migrate toward the red pulp of the spleen. There was a virtual absence of Gr-1ϩ cells in the germinal centers (B cell-rich zones). 2090 TRAUMA-INDUCED CD11bϩ/Gr-1ϩ CELLS SUPPRESS T CELL FUNCTIONS

FIGURE 8. Morphology of CD11bϩ/Gr-1ϩ cells isolated from the spleen of mice undergoing laparotomy (traumatic stress) or anesthesia only (control). CD11bϩ/Gr-1ϩ cells were enriched from the spleen (see Materials and Methods, purity ϭ 98%). Cytospin slides were prepared and stained using the LeukoStat Stain kit. Cells were identified as a mixture of mature neutrophils with characteristic lobular-shaped nuclei, as well as immature myeloid cells with a ring-shaped nucleus. Magnification ϫ100. CD11bϩ/Gr-1ϩ cells morphologically show ringed-shaped nuclei with lobulations. markers appeared to slowly decrease and migrate toward the red Thus, these data demonstrate that trauma-induced CD11bϩ/ pulp of the spleen. There was a virtual absence of Gr-1ϩ cells in Gr-1ϩ cells are closely related phenotypically to control CD11bϩ/ the germinal centers (B cell-rich zones). These data render support Gr-1ϩ cells. CD11bϩ/Gr1ϩ cells induced by traumatic stress are to the hypothesis that trauma-induced CD11bϩ/Gr-1ϩ cells could not lymphoid cells because they do not express any significant be playing a regulatory role on T cell function. levels of lymphoid markers (Table I). We conclude that trauma- ϩ ϩ ϩ ϩ induced CD11b /Gr-1 cells are a heterogeneic population of im- Morphological characterization of CD11b /Gr-1 cells mature myeloid precursor cells in as much as they express CD34, As shown in Fig. 8, CD11bϩ/Gr-1ϩ cells represented a mixture of CD16/32, F4/80, CD80, and CD86 markers. Trauma-induced ϩ ϩ myeloid cells in varying stages of differentiation. Trauma-induced CD11b /GR1 cells do not express markers of mature myeloid immature CD11bϩ/Gr-1ϩ cells exhibited a neutrophil-like mor- cells such as DEC 205, NK 1.1, CD36, CD13, and CD68. Inter- ϩ ϩ phology, suggesting a granulopoietic response. This type of mor- estingly, control CD11b /Gr-1 cells that are phenotypically re- ϩ ϩ phology appears similar to that described previously for immature lated to trauma-induced CD11b /Gr1 cells do not express ϩ ϩ myeloid cells in murine models of cancer and halogenated aro- ARG1. To our knowledge, these CD11b /Gr-1 cells have not matic hydrocarbon toxicity (20). The morphological characteristics been described previously in models of traumatic stress. ϩ ϩ of the cells were similar in CD11b /Gr-1 cells from control mice ϩ ϩ and mice subjected to traumatic stress. CD11b /Gr-1 cells suppress T cell proliferation ϩ ϩ ϩ ϩ We performed cocultures of naive T cells and CD11b /Gr-1 CD11b /Gr-1 splenocytes are myeloid precursor cells by cells obtained from animals subjected to traumatic stress or con- phenotype trols using Transwell chambers to determine the inhibitory poten- ϩ ϩ We then further determined the phenotypical characteristics of tial of trauma-induced CD11b /Gr-1 cells. T cells from control trauma-induced CD11bϩ/Gr-1ϩ cells. CD11bϩ/Gr-1ϩ cells har- syngeneic mice stimulated with anti-CD3 and anti-CD28 Abs and ϩ ϩ vested from controls were compared with CD11bϩ/Gr-1ϩ cells cocultured in the presence of trauma-induced CD11b /Gr-1 cells harvested from mice undergoing traumatic stress. We also compared changes in phenotypical characteristics of trauma-induced CD11bϩ/Gr-1ϩ cells across time. There was a significant accumulation of CD11bϩ/Gr-1ϩ cells after traumatic stress, increasing from 2 Ϯ 1% of all splenocytes at baseline to 7.8% of cells by 6 h and reaching a peak of 15 Ϯ 4% by 12 h (Fig. 9). Thus, at one point, one CD11bϩ/Gr-1ϩ cells constituted one of seven splenic cells. We then studied the expression of several cell surface molecules, comparing control and trauma-induced CD11bϩ/Gr-1ϩ cells. Neither control nor trauma-induced CD11bϩ/Gr-1ϩ cells significantly expressed markers of lymphocytes or mature macrophages, DCs, granulocytes, or NK cells such as CD3, CD19, B220, CD8␣, NK1.1, CD14, CD11c, CD40, DEC-205, CD36, CD68, CD13, or CD31. Trauma-induced CD11bϩ/Gr-1ϩ cells expressed molecules of MHC class I (89 Ϯ 7%), MHC class II (33 Ϯ 9%), and myeloid hemopoietic precursor marker CD34 (38 Ϯ 5%) (Ta- ble I). These cells also were CD16/32 positive (82 Ϯ 5%) and FIGURE 9. Coexpression of Gr-1 and CD11b markers by splenocytes express low levels of F4/80 marker (27 Ϯ 9%). The myeloid cell ϩ ϩ from control and trauma spleen. At different time points after surgical progenitor CD131 marker was not detected in CD11b /Gr-1 stress, splenocytes were harvested and stained with anti-CD11b (FITC) and cells harvested from controls or in mice subjected to traumatic anti-Gr-1 (PE) Abs and examined by FACS. At time 0, there is a paucity stress. Other markers studied were inconsistently expressed and of CDllbϩ/Gr-1ϩ cells (2%). At time intervals of 6, 12, 24, 48, and 72 h, included costimulatory molecules CD80 (17 Ϯ 7%), CD86 the percentage of CDllbϩ/Gr-1ϩ cells increase to 8, 15, 5, 12, and 11%, (23 Ϯ 9%). respectively (p Ͻ 0.05). The Journal of Immunology 2091

Table I. CD11bϩ/Gr-1ϩ cells express markers of immature myeloid cells (ﬂow cytometry data)a MHC I MHC II CD14 CD80 CD86 CD40 CD-205 CD8a CD11c CD34 CD131 CD36 CD13 F4/80 CD68 CD16/32 DEC-205 NK1.1 CD4 CD3 CD19 B220 CD31

Control CD11 bϩ Ϫ Low Ϫ Low Low ϪϪϪLow ϪϪϪϪLow Ϫ Low ϪϪϪϪϪϪLow Trauma CD11bϩϩLow Ϫ Low Low Low ϪϪLow Low ϪϪϪLow Ϫ Low ϪϪϪϪϪϪLow

a Negative (Ϫ) signs denote the absence of significant flow cytrometric levels of the phenotype studied. exhibited a significant decrease in proliferation as measured by Taken together, these data demonstrate that trauma-induced [3H]thymidine incorporation, compared with cells cocultured with CD11bϩ/Gr-1ϩ cells are capable of exerting a T cell suppressive control CD11bϩ/Gr-1ϩ cells or cultured alone (Fig. 10A). The effect, which is mediated by ARG1. ϩ most significant suppressive effect of trauma-induced CD11b / ϩ ϩ Gr-1ϩ cells on T cell proliferation was observed at 1:16 effector: Trauma-induced CD11b /Gr-1 cells suppress IL-2 production target (E:T) ratio with a two-fold inhibition of T cell proliferation by T cells (30,944 Ϯ 3443 for controls vs 13,078 Ϯ 794 cpm for trauma; p Ͻ IL-2 is a well-studied cytokine that is essential for T cell activa- 0.05). The arginase antagonist nor-NOHA restored T cell prolif- tion. Decreased IL-2 production is characteristic of traumatic ϩ ϩ eration caused by trauma-induced CD11b /Gr-1 cells (Fig. 10B). stress. To determine whether CD11bϩ/Gr-1ϩ cells isolated from Equally, supplementation of culture medium with an additional 1.2 mice subjected to traumatic stress were suppressing IL-2 produc- ϩ ϩ mM L-arginine abrogated the suppressive effect of CD11b /Gr-1 tion, we measured IL-2 accumulation in culture medium of T cell: cells on T cell proliferation (data not shown). CD11bϩ/Gr-1ϩ cell cocultures. Appropriate controls were performed using CD11bϩ/Gr-1ϩ cells harvested from nontraumatized

FIGURE 10. A and B, Trauma CD11bϩ/Gr-1ϩ cells suppress T cell proliferation due to high arginase activity. Naive T cells were stimulated with anti-CD3 plus anti-CD28 Abs and cocultured through Transwell chamber at different ratios with CD11bϩ/Gr-1ϩ cells induced by traumatic FIGURE 11. A and B, IL-2 accumulation in medium at 12 h of coin- stress and harvested after 24 h (A). CD11bϩ/Gr-1ϩ cells obtained from cubation of T cells with CD11bϩ/Gr-1ϩ cells. CD11bϩ/Gr-1ϩ cells ob- control mouse (anesthesia only) served as controls. At E:T ratio 1:16, trau- tained from mouse spleen 24 h after traumatic stress inhibit anti-CD3 ϩ matic stress-induced CD11bϩ/Gr-1ϩ cells suppress T cell proliferation. anti-CD28 Abs stimulated IL-2 production by T cells (A). CD11bϩ/Gr-1ϩ Importantly, this suppressive effect was prevented by adding nor-NOHA cells isolated from mice undergoing anesthesia only served as controls. T (an arginase antagonist), suggesting that CD11bϩ/Gr-1ϩ cells affect T cell cells were obtained from naive mouse. Nor-NOHA increased IL-2 produc- proliferation through arginase (B). Supplementation of medium with 1.2 tion to that of control levels (456 pg per ml per 0.2 M cells) as well as mM L-arginine also prevents suppression of T cell proliferation. T cell supplementation of medium by 1.2 mM L-arginine. B, The concentration of proliferation was measured in triplicates by [3H]thymidine incorporation IL-2 in cell-free supernatant of cultures was determined by p70 ELISA. after 16-h pulse and expressed in cpm. Results are given as mean Ϯ SEM Data are representative of three independent experiments and expressed as p Ͻ 0.05 ,ء ,from two representative independent experiments. mean Ϯ SEM 2092 TRAUMA-INDUCED CD11bϩ/Gr-1ϩ CELLS SUPPRESS T CELL FUNCTIONS

FIGURE 13. Control and trauma CD11bϩ/Gr-1ϩ cells poorly stimulate naive allogeneic T cell proliferation. 102–105 control or trauma-induced CD11bϩ/Gr-1ϩ cells (effectors) were added to 2 ϫ 105 naive allogeneic T cells per well in triplicate in 96-well round-bottom culture plates and incubated for 96 h in 200 ␮lof150␮M L-arginine medium at 37°C in a

humidiﬁed 5% CO2 atmosphere. T cell proliferation was measured in triplicates by [3H]thymidine incorporation after 16-h pulse and expressed in cpm. Results are given as mean Ϯ SEM from two independent experiments. At E:T ratios 1:1 and 1:4, both control and traumatic stress-induced CD11bϩ/Gr-1ϩ cells stimulate T cell proliferation, although not as strongly as DCs. Importantly, control CD11bϩ/Gr-1ϩ cells at 1:1 ratios are better at stimulating T cell proliferation compared to trauma-induced CD11bϩ/ Gr-1ϩ cells.

FIGURE 12. Traumatic stress-induced CD11bϩ/Gr-1ϩ cells suppress the TCR ␨-chain expression by T cells. Naive T cells were stimulated with ϩ ϩ ␨ anti-CD3 plus anti-CD28 Abs and cocultured with CD11b /Gr-1 cells TCR -chain expression in T cells stimulated with anti-CD3 and harvested after 24 h from control mice and mice subjected to traumatic anti-CD28 Abs from 59 Ϯ 14% in controls to 20 Ϯ 7% in T cells ϩ ϩ stress. CD11bϩ/Gr-1ϩ cells obtained from mice undergoing laparotomy cultured with trauma-induced CD11b /Gr-1 cells ( p Ͻ 0.05) inhibited TCR ␨-chain expression in 4-fold (p Ͻ 0.05). The use of nor- (Fig. 12). Once again, the suppressive effect caused by trauma- ϩ ϩ NOHA or adding of 1.2 mM L-arginine was associated with restoration of induced CD11b /Gr-1 cells was prevented by the addition of ␨ ϩ ϩ TCR -chain expression. No effects of control CD11b /Gr-1 cells on nor-NOHA (Fig. 12) or 1.2 mM L-arginine. This observation fur- ␨ -chain expression by T cells were observed. Data represent one of three ther demonstrates the important suppressive role of ARG1. independent experiments. ϩ ϩ Taken together, trauma-induced CD11b /Gr-1 cells appear to be capable of reproducing at least some of the characteristic ob- mice. T cells (obtained from nontraumatized mice) were stimu- servations of T cell suppression observed after traumatic stress. lated with anti-CD3 and anti-CD28 Abs and cocultured for 12 h Furthermore, our work demonstrates that ARG1, probably through with irradiated CD11bϩ/Gr-1ϩ cells harvested from mice sub- a mechanism of arginine depletion, may be the main mechanism ϩ ϩ jected to traumatic stress or from control animals. Fig. 11A dem- by which trauma-induced CD11b /Gr-1 cells cause T cell onstrates up to a 76% inhibition in IL-2 accumulation for T cells suppression. cocultured with trauma-induced CD11bϩ/Gr-1ϩ cells, compared Ϯ ϫ 5 with controls (114 6 pg/ml per 2 10 cells per 12 h of IL-2 ϩ ϩ vs 461 Ϯ 78 pg/ml per 2 ϫ 105 cells per 12 h of IL-2 in control) CD11b /Gr-1 cells slightly stimulate proliferation of at E:T ratio 1:16 ( p Ͻ 0.01). Importantly, these data are in agree- allogeneic naive T cells ment with our results demonstrating suppression of T cells prolif- We then investigated the potential Ag-presenting capacity of eration by CD11bϩ/Gr-1ϩ cells after traumatic stress (Fig. 9A). CD11bϩ/Gr-1ϩ cells. To answer this question, we performed a The addition of nor-NOHA or supplementation of medium with MLR using naive allogeneic T cells as targets. DCs were used as ϩ 1.2 mM L-arginine restored cytokine production (Fig. 11B). These positive controls. Pilot experiments demonstrated that CD11b / data suggest that trauma-induced CD11bϩ/Gr-1ϩ cells may ex- Gr-1ϩ cells slightly stimulate naive T cells proliferation and this plain the decrease in IL-2 production in T cells after trauma. effect is ratio dependent (Fig. 13). The highest T cell proliferation (29208 Ϯ 1355 and 26422 Ϯ 1325 cpm) was observed at 1:1 ϩ ϩ ␨ Trauma-induced CD11b /Gr-1 cells inhibit TCR -chain and 1:4 E:T ratios, respectively, and then linearly decreased expression of T cells down to 1:64 E:T ratio. No signiﬁcant stimulatory effect was To investigate the effect of CD11bϩ/Gr-1ϩ cells on T lymphocyte observed at lower E:T ratios. This ﬁnding suggests that imma- TCR ␨-chain expression, we performed cocultures of naive T cells ture myeloid suppressor CD11bϩ/Gr-1ϩ cells can stimulate na- and CD11bϩ/Gr-1ϩ cells in Transwells at E:T ratio 1:1. T cells ive T cells through the expression of MHC class I and II mol- cultured in the absence of CD11bϩ/Gr-1ϩ cells were used as con- ecules, CD80, and CD86. However, CD11bϩ/Gr-1ϩ cells are trols. Trauma-induced CD11bϩ/Gr-1ϩ cells strongly suppressed three times less potent than DCs. The Journal of Immunology 2093

Discussion systemic use of the ␤-adrenergic blocker propranolol in a model of T cell dysfunction after trauma appears to be the result of T cell- trauma (29). Trauma also induces the expression of cytokines such monocyte interactions being responsible for increased susceptibil- as IL-4, IL-6, IL-10, and IL-13, all known to induce arginase in the ity to infections (21, 22). T cell dysfunction is characterized by RAW 264.7 myeloid cell line. Prostaglandins also are released decreased production of IL-2, IFN-␥, and loss of TCR ␨-chain after trauma and also may be responsible for the induction of arginase after trauma. Sorting out the roles played by these possible expression (5, 23, 24). Preventing or reversing immune dysfunc- ϩ ϩ tion after trauma should result in better patient outcomes and candidate substances in the induction of ARG1 in CD11b /Gr-1 cells after trauma will be an important goal for future work. It is decreased cost. ϩ ϩ Trauma is associated with a significant reduction in circulating interesting that we observed a second peak of CD11b /Gr-1 cells arginine that reflects a well-known state of arginine deficiency (9, occurring 48 h after trauma. Incidentally, we also have observed a 25). The mechanisms and biological consequences of arginine de- secondary peak in arginase activity 4 days after severe trauma in ficiency have been poorly understood. In an effort to gain further humans. The mechanisms behind this second peak are currently unknown, though we speculate that delayed production of sub- understanding of the changes in arginine metabolism that occur ϩ after trauma, our group has developed a mouse model of moderate stances that induce the proliferation of CD11b cells may be surgical trauma, whose alterations closely mimic those observed in implicated. human surgical patients and trauma victims. Dietary arginine supplementation is associated with the restora- Increased destruction of arginine by the enzyme ARG1 has been tion of T cell counts in surgical patients and has been associated with a decrease in postoperative infection rates (30). These clinical recently described as a mechanism of T cell regulation in diseases observations suggested to us that impaired T cell function after such as cancer (26). ARG1 is not expressed in immune tissues trauma could be caused by arginine deficiency. We have thor- under resting conditions, although it is induced within hours of a oughly tested this hypothesis in vitro using mouse T lymphocytes physical injury. Several years ago, our group detected increased and T cell lines, identifying key molecular effects of arginine de- arginase activity and ARG1 expression in human peripheral mono- ficiency (6). Withholding L-arginine from the culture medium nuclear cells after trauma or surgery and also in a mouse model of ϩ ϩ leads to a significant decrease in the expression of the TCR and the surgical trauma (9, 12). This study identifies the CD11b /Gr-1 TCR ␨-chain peptide (7). Loss of ␨-chain has been described in immature myeloid cells that exclusively express ARG1 after trau- cancer and also in trauma. We demonstrate in this study that we matic stress (Figs. 1–3). It is interesting that ARG1 is not induced can reproduce the loss of the TCR ␨-chain, suppression of T cell in other cell types by trauma and clearly not expressed in lymphoid proliferation, and IL-2 production through the coculture of T lym- cells (Figs. 1 and 2). Similar to reports presented in models of ϩ ϩ phocytes and trauma-induced CD11b /Gr-1 cells (Fig. 12). Fur- cancer, trauma-induced cells expressing ARG1 are of myeloid or- ϩ thermore, we demonstrate that trauma-induced immature CD11b / igin and express CD11b and Gr-1 markers (27 and Table I). These ϩ Gr-1 cells exhibit increased L-arginine uptake (Fig. 5) and cells exhibit lobular and ring-shaped nuclei that are characteristic significantly deplete L-arginine from the culture medium (Fig. 6A). of neutrophils and immature myeloid cells (Fig. 8). However, as Trauma-induced immature CD11bϩ/Gr-1ϩ cells rapidly accumu- confirmed by phenotype analysis, these cells are not mature neu- late in the spleen within hours of induction of traumatic stress and trophils or granulocytes, because they do not express any signifi- colocalize with T cells in specific zones of the spleen, thus sug- cant levels of CD68 or CD13 markers. In addition, in our prelim- gesting an important interaction with them (Fig. 7). inary experiments, we did not detect significant levels of reactive These findings support the hypothesis that arginine depletion by oxygen species production, again suggesting that these cells are myeloid cells is a novel mechanism of T cell regulation. The work not granulocytes (data not shown). Interestingly, ARG1-express- presented in this study provides new avenues for the development ing myeloid cells have varied morphological characteristics, de- of effective mechanisms of overcoming T cell dysfunction after pending on the various models described. For example, Choi et al. trauma. These include dietary strategies for arginine replacement, (20), depicts a cell that is very similar to the one described by us. prevention of ARG1 induction, and pharmacologic arginase block- However, Rodriguez et al. (28) reports the virtual invasion of an ϩ Ϫ ade. Our work presents a reliable trauma model in which to test arginase-expressing CD11b /Gr-1 cell into tumors, which ex- these possible strategies. hibit characteristics of more mature myeloid cells. In contrast, ϩ Ϫ CD11b /Gr-1 cell subpopulations observed in trauma did not Acknowledgments express any significant arginase activity (Fig. 3). ϩ ϩ We thank Dr. Sidney Morris, Jr., and Dr. Michael R. Shurin (University of Trauma-induced CD11 /Gr-1 cells exhibit significant differ- Pittsburgh, Pittsburgh, PA) for constructive comments during our work and ences from other myeloid cells reported. For example, they ex- for providing anti-mouse-ARG1 Ab; Dr. Angus Thomson for guidance in presses only low levels F4/80 or CD31 (Table I). There also are the isolation and characterization of immature myeloid cells; and dramatic temporal differences in the appearance of trauma-induced Dr. Rosemary Hoffman for assistance in the design of T cell coculture immature CD11bϩ/Gr-1ϩ cells (which are observed within hours experiments. We also thank Dr. Augusto Ochoa for inspiration and en- of a trauma, Figs. 7 and 9) and those observed in cancer, which couragement to continue this work. only occurs after days or weeks of tumor implantation. Finally, the magnitude of the increase in ARG1 expression appears to be far Disclosures higher after trauma, compared with cancer models. These obser- J.B.O. has a patent application on the possible function of arginase in immune suppression, has received grants from the National Institutes of vations imply significant differences in the type of cell and mech- Health and the Pittsburgh Society Foundation, and has consultant agree- anisms of induction of ARG1 among the different disease ments with Ross-Abbott Laboratories and Novartis Pharmaceutical Corp. processes. The mechanisms behind the induction of ARG1 and the appear- ϩ ϩ References ance and accumulation of CD11b /Gr-1 cells have been only 1. Sauaia, A., F. A. Moore, E. E. Moore, K. S. Moser, R. Brennan, R. A. Read, and partially studied. We reported previously that arginase activity P. T. Pons. 1995. Epidemiology of trauma deaths: a reassessment. J. Trauma 38: 185–193. could be induced by catecholamines and demonstrated a signifi- 2. Messingham, K. A., D. E. Faunce, and E. J. Kovacs. 2002. Alcohol, injury, and cant “blunting” of arginase induction in splenic tissue with the cellular immunity. Alcohol 28: 137–149. 2094 TRAUMA-INDUCED CD11bϩ/Gr-1ϩ CELLS SUPPRESS T CELL FUNCTIONS

3. Cheadle, W. G., M. Mercer-Jones, M. Heinzelmann, and H. C. Polk, Jr. 1996. 17. Konarska, L., and L. Tomaszewski. 1986. A simple quantitative micromethod or Sepsis and septic complications in the surgical patient: who is at risk? Shock 6 arginase assay in blood spots dried on filter paper. Clin. Chim. Acta 154: 7–17. (Suppl. 1): S6–S9. 18. Ochoa, J. B., A. C. Bernard, S. K. Mistry, S. M. J. Morris, P. L. Figert, 4. De, A. K., K. M. Kodys, J. Pellegrini, B. Yeh, R. K. Furse, P. Bankey, and M. E. Maley, B. J. Tsuei, B. R. Boulanger, and P. A. Kearney. 2000. Trauma C. L. Miller-Graziano. 2000. Induction of global anergy rather than inhibitory increases extrahepatic arginase activity. Surgery 127: 419–426. Th2 lymphokines mediates posttrauma T cell immunodepression. Clin. Immunol. 19. Gabrilovich, D. I., M. P. Velders, E. M. Sotomayor, and W. M. Kast. 2001. 96: 52–66. ϩ Mechanism of immune dysfunction in cancer mediated by immature Gr-1 my- 5. Ichihara, F., K. Kono, T. Sekikawa, and Y. Matsumoto. 1999. Surgical stress eloid cells. J. Immunol. 166: 5398–5406. induces decreased expression of signal-transducing ␨ molecules in T cells. Eur. 20. Choi, J. Y., J. A. Oughton, and N. I. Kerkvliet. 2003. Functional alterations in Surg. Res. 31: 138–146. ϩ ϩ 6. Ochoa, J. B., J. Strange, P. Kearney, G. Gellin, E. Endean, and E. Fitzpatrick. CD11b Gr-1 cells in mice injected with allogeneic tumor cells and treated with 2001. Effects of L-arginine on the proliferation of T lymphocyte subpopulations. 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. Immunopharmacol. 3: 553–570. J. Parenter. Enteral Nutr. 25: 23–29. 21. De, A. K., K. Kodys, J. C. Puyana, G. Fudem, J. Pellegrini, and 7. Rodriguez, P. C., A. H. Zea, K. S. Culotta, J. Zabaleta, J. B. Ochoa, and C. L. Miller-Graziano. 1997. Only a subset of trauma patients with depressed A. C. Ochoa. 2002. Regulation of T cell receptor CD3␨ chain expression by mitogen responses have true T cell dysfunctions. Clin. Immunol. Immunopathol. L-arginine. J. Biol. Chem. 277: 21123–21129. 82: 73–82. 8. Taheri, F., J. B. Ochoa, Z. Faghiri, K. Culotta, H. J. Park, M. S. Lan, A. H. Zea, 22. De, A. K., K. Laudanski, and C. L. Miller-Graziano. 2003. Failure of monocytes and A. C. Ochoa. 2001. L-Arginine regulates the expression of the T-cell recep- of trauma patients to convert to immature dendritic cells is related to preferential tor-␨ chain (CD3␨) in Jurkat cells. Clin. Cancer Res. 7 (Suppl. 3): S958–S965. macrophage-colony-stimulating factor-driven macrophage differentiation. J. Im- 9. Ochoa, J. B., A. C. Bernard, W. E. O’Brien, M. M. Griffen, M. E. Maley, munol. 170: 6355–6362. A. K. Rockich, B. J. Tsuei, B. R. Boulanger, P. A. Kearney, and J. S. Morris, Jr. 23. Faist, E., C. Schinkel, and S. Zimmer. 1996. Update on the mechanisms of im- 2001. Arginase I expression and activity in human mononuclear cells after injury. mune suppression of injury and immune modulation. World J. Surg. 20: Ann. Surg. 233: 393–399. 454–459. 10. Daly, J. M., M. D. Lieberman, J. Goldfine, J. Shou, F. Weintraub, E. F. Rosato, and P. Lavin. 1992. Enteral nutrition with supplemental arginine, RNA, and ome- 24. Ertel, W., E. Faist, C. Nestle, L. Hueltner, M. Storck, and F. W. Schildberg. 1990. ga-3 fatty acids in patients after operation: immunologic, metabolic, and clinical Kinetics of interleukin-2 and interleukin-6 synthesis following major mechanical outcome. Surgery 112: 56–67. trauma. J. Surg. Res. 48: 622–628. 11. Heyland, D. K., F. Novak, J. W. Drover, M. Jain, X. Su, and U. Suchner. 2001. 25. Morris, S. M., Jr. 2004. Enzymes of arginine metabolism. J. Nutr. 134 (Suppl. Should immunonutrition become routine in critically ill patients? A systematic 10): S2743–S2747. review of the evidence. J. Am. Med. Assoc. 286: 944–953. 26. Bronte, V., and P. Zanovello. 2005. Regulation of immune responses by L-argi- 12. Bernard, A. C., S. K. Mistry, S. M. Morris, Jr., W. E. O’Brien, B. J. Tsuei, nine metabolism. Nat. Rev. Immunol. 5: 641–654. M. E. Maley, L. A. Shirley, P. A. Kearney, B. R. Boulanger, and J. B. Ochoa. 27. Kusmartsev, S. A., Y. Li, and S. H. Chen. 2000. Gr-1ϩ myeloid cells derived 2001. Alterations in arginine metabolic enzymes in trauma. Shock 15: 215–219. from tumor-bearing mice inhibit primary T cell activation induced through CD3/ 13. Bronte, V., P. Serafini, A. Mazzoni, D. M. Segal, and P. Zanovello. 2003. L- CD28 costimulation. J. Immunol. 165: 779785. Arginine metabolism in myeloid cells controls T-lymphocyte functions. Trends 28. Rodriguez, P. C., D. G. Quiceno, J. Zabaleta, B. Ortiz, A. H. Zea, M. B. Piazuelo, Immunol. 24: 302–306. A. Delgado, P. Correa, J. Brayer, E. M. Sotomayor, et al. 2004. Arginase I 14. Rodriguez, P. C., A. H. Zea, J. DeSalvo, K. S. Culotta, J. Zabaleta, production in the tumor microenvironment by mature myeloid cells inhibits T- D. G. Quiceno, J. B. Ochoa, and A. C. Ochoa. 2003. L-Arginine consumption by cell receptor expression and antigen-specific T-cell responses. Cancer Res. 64: macrophages modulates the expression of CD3-␨ chain in T lymphocytes. J. Im- 5839–5849. munol. 171: 1232–1239. 15. Bradford, M. 1976. A rapid and sensitive method for the quantitation of micro- 29. Bernard, A. C., E. A. Fitzpatrick, M. E. Maley, G. L. Gellin, B. J. Tsuei, gram quantities of protein utilizing the principle of protein-dye binding. Anal. W. A. Arden, B. R. Boulanger, P. A. Kearney, and J. B. Ochoa. 2000. ␤-adre- Biochem. 72: 248–254. noceptor regulation of macrophage arginase activity. Surgery 127: 412–418. 16. Morris, S. M., Jr., D. Kepka-Lenhart, and L. C. Chen. 1998. Differential regula- 30. Daly, J. M., J. Reynolds, A. Thom, L. Kinsley, M. Dietrick-Gallagher, J. Shou, tion of arginases and inducible nitric oxide synthase in murine macrophage cells. and B. Ruggieri. 1988. Immune and metabolic effects of arginine in the surgical Am. J. Physiol. 275: E740–E747. patient. Ann. Surg. 208: 512–522.