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 ob- served 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 dysfunc- tion 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. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2086 TRAUMA-INDUCED CD11bϩ/Gr-1ϩ CELLS SUPPRESS T CELL FUNCTIONS 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 main- tained 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).