Biometric Profiling to Quantify Lung Injury Through Ex Vivo Lung Perfusion Following Warm Ischemia.

Standard physiologic assessment parameters of donor lung grafts may not accurately reflect lung injury or quality. A biometric profile of ischemic injury could be identified as a means to assess the quality of the donor allograft. We sought to identify a biometric profile of lung ischemic injury assessed during ex vivo lung perfusion (EVLP). A rat model of lung donation after circulatory death (DCD) warm ischemic injury with subsequent EVLP evaluation was utilized. We did not observe a significant correlation between the classical physiological assessment parameters and the duration of the ischemic. In the perfusate, solubilized lactate dehydrogenase (LDH) as well as hyaluronic acid (HA) significantly correlated with duration of ischemic injury and length of perfusion ( p < 0.05). Similarly, in perfusates, the endothelin-1 (ET-1) and Big ET-1 correlated ischemic injury ( p < 0.05) and demonstrated a measure of endothelial cell injury. In tissue protein expression, heme oxygenase-1 (HO-1), angiopoietin 1 (Ang-1), and angiopoietin 2 (Ang-2) levels were correlated with the duration of ischemic injury ( p < 0.05). Cleaved caspase-3 levels were significantly elevated at 90 and 120 minutes ( p < 0.05) demonstrating increased apoptosis. A biometric profile of solubilized and tissue protein markers correlated with cell injury is a critical tool to aid in the evaluation of lung transplantation, as accurate evaluation of lung quality is imperative and improved quality leads to better results. http://links.lww.com/ASAIO/B49.

Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples.

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1ß, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFß did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.

Electrical Impedance as a Noninvasive Metric of Quality in Allografts Undergoing Normothermic Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) increases the pool of suitable organs for transplant by facilitating assessment and repair at normothermia, thereby improving identification of quality of marginal organs. However, there exists no current objective approach for assessing total organ edema. We sought to evaluate the use of electrical impedance as a metric to assess total organ edema in lungs undergoing EVLP. Adult porcine lungs (40 kg) underwent normothermic EVLP for 4 hours. To induce varying degrees of lung injury, the allografts were perfused with either Steen, a modified cell culture media, or 0.9% normal saline. Physiologic parameters (peak airway pressure and compliance), pulmonary artery and left atrial blood gases, and extravascular lung water measurements were evaluated over time. Wet-to-dry ratios were evaluated postperfusion. Modified Murray scoring was used to calculate lung injury. Impedance values were associated with lung injury scores ( p = 0.007). Peak airway pressure ( p = 0.01) and PaO 2 /FiO 2 ratios ( p = 0.005) were both significantly associated with reduced impedance. Compliance was not associated with impedance ( p = 0.07). Wet/dry ratios were significantly associated with impedance and Murray Scoring within perfusion groups of Steen, Saline, and Modified Cell Culture ( p = 0.0186, 0.0142, 0.0002, respectively). Electrical impedance offers a noninvasive modality for measuring lung quality as assessed by tissue edema in a porcine model of normothermic EVLP. Further studies evaluating the use of impedance to assess organ edema as a quality marker in human clinical models and abdominal organs undergoing ex vivo perfusion warrant investigation.

PolyADP-Ribosylation of NFATc3 and NF-κB Transcription Factors Modulate Macrophage Inflammatory Gene Expression in LPS-Induced Acute Lung Injury.

Pulmonary macrophages play a critical role in the recognition of pathogens, initiation of host defense via inflammation, clearance of pathogens from the airways, and resolution of inflammation. Recently, we have shown a pivotal role for the nuclear factor of activated T-cell cytoplasmic member 3 (NFATc3) transcription factor in modulating pulmonary macrophage function in LPS-induced acute lung injury (ALI) pathogenesis. Although the NFATc proteins are activated primarily by calcineurin-dependent dephosphorylation, here we show that LPS induces posttranslational modification of NFATc3 by polyADP-ribose polymerase 1 (PARP-1)-mediated polyADP-ribosylation. ADP-ribosylated NFATc3 showed increased binding to iNOS and TNFα promoter DNA, thereby increasing downstream gene expression. Inhibitors of PARP-1 decreased LPS-induced NFATc3 ribosylation, target gene promoter binding, and gene expression. LPS increased NFAT luciferase reporter activity in lung macrophages and lung tissue that was inhibited by pretreatment with PARP-1 inhibitors. More importantly, pretreatment of mice with the PARP-1 inhibitor olaparib markedly decreased LPS-induced cytokines, protein extravasation in bronchoalveolar fluid, lung wet-to-dry ratios, and myeloperoxidase activity. Furthermore, PARP-1 inhibitors decreased NF-кB luciferase reporter activity and LPS-induced ALI in NF-кB reporter mice. Thus, our study demonstrates that inhibiting NFATc3 and NF-кB polyADP-ribosylation with PARP-1 inhibitors prevented LPS-induced ALI pathogenesis.

Pegylated-Catalase Is Protective in Lung Ischemic Injury and Oxidative Stress.

BACKGROUND: Lung transplant ischemia-reperfusion injury is typified by toxic metabolites and oxygen free radicals leading to worse graft function. Catalase is an enzyme involved in oxidative-stress detoxification. We hypothesize that direct delivery of highly concentrated polyethylene glycol-catalase (PEG-CAT) during normothermic ex vivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. METHODS: To demonstrate protection, primary culture porcine endothelial cells were treated with PEG-CAT (0 to 1250 U/mL) in a model of oxidative stress (400 µM H2o2). In vivo, rat lungs were subjected to 0 hours or 1 hour of warm ischemic injury and 2 hours of EVLP with or without PEG-CAT. Perfusate was collected throughout the perfusion duration and tissue was collected at the end. Tissue and perfusate underwent analysis for markers of apoptosis and a biometric signature of lung health. RESULTS: Uptake of PEG-CAT into primary endothelial cells was demonstrated with Alexa Fluor 488-labeled PEG-CAT. Oxidatively stressed cells pretreated with PEG-CAT had significantly decreased cytotoxicity and caspase 3/7 activity and increased cell viability and cell membrane integrity. In a rat model of warm ischemia with EVLP, PEG-CAT improved allograft viability as measured by indications of cell membrane integrity (lactate dehydrogenase and hyaluronic acid), presence of vasoconstrictive peptides (endothelin-1 and big endothelin-1) released from endothelial cells, and reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling). CONCLUSIONS: In vitro and ex vivo, PEG-CAT protects against oxidative stress-induced cytotoxicity, maintains cellular metabolism, and mitigates lung ischemia-reperfusion in an experimental model. Together, these data suggest that PEG-CAT is a potential therapeutic target for donor organs at risk for ischemia-reperfusion injury.

IRAK-M Regulates Monocyte Trafficking to the Lungs in Response to Bleomycin Challenge.

Idiopathic pulmonary fibrosis is a deadly disease characterized by excessive extracellular matrix deposition in the lungs, resulting in decreased pulmonary function. Although epithelial cells and fibroblasts have long been the focus of idiopathic pulmonary fibrosis research, the role of various subpopulations of macrophages in promoting a fibrotic response is an emerging target. Healthy lungs are composed of two macrophage populations, tissue-resident alveolar macrophages and interstitial macrophages, which help to maintain homeostasis. After injury, tissue-resident alveolar macrophages are depleted, and monocytes from the bone marrow (BM) traffic to the lungs along a CCL2/CCR2 axis and differentiate into monocyte-derived alveolar macrophages (Mo-AMs), which is a cell population implicated in murine models of pulmonary fibrosis. In this study, we sought to determine how IL-1R-associated kinase-M (IRAK-M), a negative regulator of TLR signaling, modulates monocyte trafficking into the lungs in response to bleomycin. Our data indicate that after bleomycin challenge, mice lacking IRAK-M have decreased monocyte trafficking and reduced Mo-AMs in their lungs. Although IRAK-M expression did not regulate differences in chemokines, cytokines, or adhesion molecules associated with monocyte recruitment, IRAK-M was necessary for CCR2 upregulation following bleomycin challenge. This finding prompted us to develop a competitive BM chimera model, which demonstrated that expression of BM-derived IRAK-M was necessary for monocyte trafficking into the lung and for subsequent enhanced collagen deposition. These data indicate that IRAK-M regulates monocyte trafficking by increasing the expression of CCR2, resulting in enhanced monocyte translocation into the lung, Mo-AM differentiation, and development of pulmonary fibrosis.

[D-Ala2, D-Leu5] Enkephalin Improves Liver Preservation During Normothermic Ex Vivo Perfusion.

BACKGROUND: Meeting the metabolic demands of donor livers using normothermic ex vivo liver perfusion (NEVLP) preservation technology is challenging. The delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has been reported to decrease the metabolic demand in models of ischemia and cold preservation. We evaluated the therapeutic potential of DADLE by investigating its ability to protect against oxidative stress and hepatic injury during normothermic perfusion. MATERIALS AND METHODS: Primary rat hepatocytes were used in an in vitro model of oxidative stress to determine the minimum dose of DADLE needed to induce protection and the mechanisms associated with protection. NEVLP was then used to induce injury in rat livers and determine the effectiveness of DADLE in preventing liver injury. RESULTS: In hepatocytes, DADLE was protective against oxidative stress and led to a decrease in phosphorylation of JNK and p38. Naltrindole, a δ-opioid receptor antagonist, blocked this effect. DADLE also activated the PI3K/Akt signaling pathway, and PI3K/Akt inhibition decreased the protective effects of DADLE treatment. In addition, DADLE treatment during NEVLP resulted in lower perfusate alanine aminotransferase and tissue malondialdehyde and better tissue adenosine triphosphate and glutathione. Furthermore, perfusion with DADLE compared with perfusate alone preserved tissue architecture. CONCLUSIONS: DADLE confers protection against oxidative stress in hepatocytes and during NEVLP. These data suggest that the mechanism of protection involved the prevention of mitochondrial dysfunction by opioid receptor signaling and subsequent increased expression of prosurvival/antiapoptotic signaling pathways. Altogether, data suggest that opioid receptor agonism may serve as therapeutic target for improved liver protection during NEVLP.

Intrahepatic Delivery of Pegylated Catalase Is Protective in a Rat Ischemia/Reperfusion Injury Model.

BACKGROUND: Ischemia/reperfusion injury (IRI) can occur during liver surgery. Endogenous catalase is important to cellular antioxidant defenses and is critical to IRI prevention. Pegylation of catalase (PEG-CAT) improves its therapeutic potential by extending plasma half-life, but systemic administration of exogenous PEG-CAT has been only mildly therapeutic for hepatic IRI. Here, we investigated the protective effects of direct intrahepatic delivery of PEG-CAT during IRI using a rat hilar clamp model. MATERIALS AND METHODS: PEG-CAT was tested in vitro and in vivo. In vitro, enriched rat liver cell populations were subjected to oxidative stress injury (H2O2), and measures of cell health and viability were assessed. In vivo, rats underwent segmental (70%) hepatic warm ischemia for 1 h, followed by 6 h of reperfusion, and plasma alanine aminotransferase and aspartate aminotransferase, tissue malondialdehyde, adenosine triphosphate, and GSH, and histology were assessed. RESULTS: In vitro, PEG-CAT pretreatment of liver cells showed substantial uptake and protection against oxidative stress injury. In vivo, direct intrahepatic, but not systemic, delivery of PEG-CAT during IRI significantly reduced alanine aminotransferase and aspartate aminotransferase in a time-dependent manner (P < 0.01, P < 0.0001, respectively, for all time points) compared to control. Similarly, tissue malondialdehyde (P = 0.0048), adenosine triphosphate (P = 0.019), and GSH (P = 0.0015), and the degree of centrilobular necrosis, were improved by intrahepatic compared to systemic PEG-CAT delivery. CONCLUSIONS: Direct intrahepatic administration of PEG-CAT achieved significant protection against IRI by reducing the volume distribution and taking advantage of the substantial hepatic first-pass uptake of this molecule. The mode of delivery was an important factor for protection against hepatic IRI by PEG-CAT.

Interferon-γ Promotes Antibody-mediated Fratricide of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

Reprogramming Nurse-like Cells with Interferon γ to Interrupt Chronic Lymphocytic Leukemia Cell Survival.

Nurse-like cells (NLCs) play a central role in chronic lymphocytic leukemia (CLL) because they promote the survival and proliferation of CLL cells. NLCs are derived from the monocyte lineage and are driven toward their phenotype via contact-dependent and -independent signals from CLL cells. Because of the central role of NLCs in promoting disease, new strategies to eliminate or reprogram them are needed. Successful reprogramming may be of extra benefit because NLCs express Fcγ receptors (FcγRs) and thus could act as effector cells within the context of antibody therapy. IFNγ is known to promote the polarization of macrophages toward an M1-like state that is no longer tumor-supportive. In an effort to reprogram the phenotype of NLCs, we found that IFNγ up-regulated the M1-related markers CD86 and HLA-DR as well as FcγRIa. This corresponded to enhanced FcγR-mediated cytokine production as well as rituximab-mediated phagocytosis of CLL cells. In addition, IFNγ down-regulated the expression of CD31, resulting in withdrawal of the survival advantage on CLL cells. These results suggest that IFNγ can re-educate NLCs and shift them toward an effector-like state and that therapies promoting local IFNγ production may be effective adjuvants for antibody therapy in CLL.

FoxO1 regulates allergic asthmatic inflammation through regulating polarization of the macrophage inflammatory phenotype.

Inflammatory monocyte and tissue macrophages influence the initiation, progression, and resolution of type 2 immune responses, and alveolar macrophages are the most prevalent immune-effector cells in the lung. While we were characterizing the M1- or M2-like macrophages in type 2 allergic inflammation, we discovered that FoxO1 is highly expressed in alternatively activated macrophages. Although several studies have been focused on the fundamental role of FoxOs in hematopoietic and immune cells, the exact role that FoxO1 plays in allergic asthmatic inflammation in activated macrophages has not been investigated. Growing evidences indicate that FoxO1 acts as an upstream regulator of IRF4 and could have a role in a specific inflammatory phenotype of macrophages. Therefore, we hypothesized that IRF4 expression regulated by FoxO1 in alveolar macrophages is required for established type 2 immune mediates allergic lung inflammation. Our data indicate that targeted deletion of FoxO1 using FoxO1-selective inhibitor AS1842856 and genetic ablation of FoxO1 in macrophages significantly decreases IRF4 and various M2 macrophage-associated genes, suggesting a mechanism that involves FoxO1-IRF4 signaling in alveolar macrophages that works to polarize macrophages toward established type 2 immune responses. In response to the challenge of DRA (dust mite, ragweed, and Aspergillus) allergens, macrophage specific FoxO1 overexpression is associated with an accentuation of asthmatic lung inflammation, whereas pharmacologic inhibition of FoxO1 by AS1842856 attenuates the development of asthmatic lung inflammation. Thus, our study identifies a role for FoxO1-IRF4 signaling in the development of alternatively activated alveolar macrophages that contribute to type 2 allergic airway inflammation.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Imipramine attenuates neuroinflammatory signaling and reverses stress-induced social avoidance.

Psychosocial stress is associated with altered immunity, anxiety and depression. Previously we showed that repeated social defeat (RSD) promoted microglia activation and social avoidance behavior that persisted for 24days after cessation of RSD. The aim of the present study was to determine if imipramine (a tricyclic antidepressant) would reverse RSD-inducedsocial avoidance and ameliorate neuroinflammatory responses. To test this, C57BL/6 mice were divided into treatment groups. One group from RSD and controls received daily injections of imipramine for 24days, following 6 cycles of RSD. Two other groups were treated with saline. RSD mice spent significantly less time in the interaction zone when an aggressor was present in the cage. Administration of imipramine reversed social avoidance behavior, significantly increasing the interaction time, so that it was similar to that of control mice. Moreover, 24days of imipramine treatment in RSD mice significantly decreased stress-induced mRNA levels for IL-6 in brain microglia. Following ex vivo LPS stimulation, microglia from mice exposed to RSD, had higher mRNA expression of IL-6, TNF-α, and IL-1ß, and this was reversed by imipramine treatment. In a second experiment, imipramine was added to drinking water confirming the reversal of social avoidant behavior and decrease in mRNA expression of IL-6 in microglia. These data suggest that the antidepressant imipramine may exert its effect, in part, by down-regulating microglial activation.

Epstein-Barr virus (EBV)-encoded dUTPase and chronic restraint induce impaired learning and memory and sickness responses.

Most adult humans have been infected with Epstein-Barr virus (EBV) and carry the latent virus. The EBV genome codes for several proteins that form an early antigen complex important for viral replication; one of these proteins is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). The EBV-encoded dUTPase can induce sickness responses in mice. Because stress can increase latent virus reactivation, we hypothesized that chronic restraint would exacerbate sickness behaviors elicited by EBV-encoded dUTPase. Male Swiss-Webster mice were injected daily for 15 days with either saline or EBV-encoded dUTPase. Additionally, half of the mice from each condition were either restrained for 3h daily or left undisturbed. Restraint stress impaired learning and memory in the passive avoidance chamber; impaired learning and memory was due to EBV-encoded dUTPase injected into restrained mice. EBV-encoded dUTPase induced sickness responses and restraint stress interacts with EBV-encoded dUTPase to exacerbate the sickness response. These data support a role for EBV-encoded dUTPase and restraint stress in altering the pathophysiology of EBV independent of viral replication.

Social stress up-regulates inflammatory gene expression in the leukocyte transcriptome via ß-adrenergic induction of myelopoiesis.

Across a variety of adverse life circumstances, such as social isolation and low socioeconomic status, mammalian immune cells have been found to show a conserved transcriptional response to adversity (CTRA) involving increased expression of proinflammatory genes. The present study examines whether such effects might stem in part from the selective up-regulation of a subpopulation of immature proinflammatory monocytes (Ly-6c(high) in mice, CD16(-) in humans) within the circulating leukocyte pool. Transcriptome representation analyses showed relative expansion of the immature proinflammatory monocyte transcriptome in peripheral blood mononuclear cells from people subject to chronic social stress (low socioeconomic status) and mice subject to repeated social defeat. Cellular dissection of the mouse peripheral blood mononuclear cell transcriptome confirmed these results, and promoter-based bioinformatic analyses indicated increased activity of transcription factors involved in early myeloid lineage differentiation and proinflammatory effector function (PU.1, NF-κB, EGR1, MZF1, NRF2). Analysis of bone marrow hematopoiesis confirmed increased myelopoietic output of Ly-6c(high) monocytes and Ly-6c(intermediate) granulocytes in mice subject to repeated social defeat, and these effects were blocked by pharmacologic antagonists of ß-adrenoreceptors and the myelopoietic growth factor GM-CSF. These results suggest that sympathetic nervous system-induced up-regulation of myelopoiesis mediates the proinflammatory component of the leukocyte CTRA dynamic and may contribute to the increased risk of inflammation-related disease associated with adverse social conditions.

ß-Adrenergic receptor mediated increases in activation and function of natural killer cells following repeated social disruption.

Natural killer (NK) cells are specialized innate lymphocytes important in the early defense against tumor and virus bearing cells. Many factors influence the immune system's effectiveness against pathogens, including stress. Social disruption (SDR) "primes" macrophages/monocytes and dendritic cells thereby enhancing their anti-microbial function. What remains unclear is whether similar responses are evident in NK cells. Current studies investigated the cellular distribution and activation/inhibitory phenotypes of NK cells in the spleen, lung, and blood of C57BL/6 male mice following SDR. Furthermore, cytolytic activity and anti-viral cytokine production of splenic NK cells were determined. Lastly, ß-adrenergic receptor (ß-AR) signaling was investigated to determine possible mechanisms behind the SDR-induced NK cell alterations. Results indicated NK cells from SDR mice have increased expression of CD16 and CD69 and reduced NKG2a and Ly49a expression on splenic CD3-/DX5+ NK cells indicative of an activated phenotype, both immediately and 14h post-SDR. Administration of propranolol (10mg/kg; non-selective ß-adrenergic receptor antagonist) was shown to block these "priming" effects at the 14h time-point. In the lung, SDR had similar effects on activation and inhibitory receptors 14h post-SDR, however no alterations were evident in the blood besides increased NK cells directly after SDR. Additionally, splenic NK cells from SDR mice had increased CD107a surface expression, cytolytic activity, and IFN-γ production was increased upon costimulation with IgG and IL-2 ex vivo. Collectively, these data suggest that social stress "primes" NK cells in the spleen and lung to be more proficient in their cytolytic and anti-viral/tumor effecter functions through ß-adrenergic receptor dependent signaling.