Activation Of CD11b Research Articles

A Dual-Role of Gu-4 in Suppressing HMGB1 Secretion and Blocking HMGB1 Pro-Inflammatory Activity during Inflammation

BackgroundHigh mobility group box 1(HMGB1) was first recognized as a nuclear protein that increased the chromatin remodeling and regulates transcription of many genes. In recent years, HMGB1 has been identified as a critical “late” pro-inflammatory mediator due to its unique secretion pattern and lethal effects in sepsis. Therefore, preventing the active release and inhibiting the pro-inflammatory activity of HMGB1 become promising strategies for the treatment of sepsis. Here, we reported the therapeutic effects of Gu-4, a lactosyl derivative, on sepsis and the underlying molecular mechanisms.Methodology/Principal FindingsIn an experimental rat model of sepsis caused by cecal ligation and puncture (CLP), Gu-4 administration prominently attenuated lung injury and improved the survival of the septic animals, which was positively correlated with the decrease of the serum HMGB1 level. Using RAW264.7 macrophage cell line, we further showed that Gu-4 significantly suppressed the lipopolysaccharide (LPS)-induced release and cytoplasmic translocation of HMGB1. Moreover, Gu-4 not only dose-dependently attenuated recombinant human (rhHMGB1)-induced production of TNF-α, IL-6, and IL-1β in THP-1 cells, but also greatly inhibited the adhesion of rhHMGB1-challenged THP-1 cells to HUVECs. Analyses of flow cytometry demonstrated that Gu-4 could effectively reduce the activation of CD11b elicited by rhHMGB1. Western blot analyses revealed that Gu-4 treatment could partially block the rhHMGB1-induced activation of ERK and NF-κB signalings. Meanwhile, CD11b knockdown also obviously attenuated the rhHMGB1-induced phosphorylations of ERK and IKKα/β.Conclusions/SignificanceTaken together, our results suggest that Gu-4 possesses a therapeutic potential in the treatment of sepsis probably via inhibiting the LPS-induced release of HMGB1 from macrophages and via suppressing the pro-inflammatory activity of HMGB1.

Magnesium Lithospermate B Reduces Inflammatory Response in a Mouse Model of Hepatic Ischemia–Reperfusion Injury

It has been well proved that acute inflammatory response and hepatocellular apoptosis contributed to the pathogenesis of liver ischemia reperfusion (IR) injury. A vast amount of research has demonstrated that magnesium lithospermate B (MLB) has potent anti-apoptosis and potential anti-inflammatory pharmacological properties. However, it has not previously been examined whether MLB can attenuate hepatic IR injury. Firstly, the optimal dose of MLB to protect against hepatic IR injury was determined using hepatic IR model in mice. Then, the effect of MLB on IR-induced inflammatory response was detected in detail. We found that MLB exhibited protective effect from the beginning of 50 mg/kg and culminated at the doses of 100 and 200 mg/kg. The alanine aminotransferase and aspartate aminotransferase levels in MLB group were markedly decreased. Severe hepatocellular swelling/necrosis, sinusoidal/vascular congestion and inflammatory cell infiltration were seen and a large number of apoptotic cells were found in the liver samples from Saline group, while minimal damage and very few apoptotic cells were noted in the samples from MLB group. Kuppfer cells infiltration, myeloperoxidase activity and mRNA level of CD11b in MLB group was significantly decreased. Serum TNF-a and IL-6, and mRNA expression of CXCL-10 and ICAM-1 was markedly decreased in the samples from MLB group. Inflammatory signaling pathway activation was largely prevented in MLB group. MLB can significantly attenuate IR-induced hepatocellular damage and hepatocellular apoptosis by preventing inflammatory signaling pathways activation, inflammatory mediators expression and macrophage and neutrophil infiltration.

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood. To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages. Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays. Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Increased Angiogenic Property Of Human Peripheral Blood Monocytes By ex Vivo Culture With c-Mpl Agonists In Hindlimb Ischemia Mouse Model

Human peripheral blood mononuclear cells (PB-MNCs) include some populations which have angiogenic properties. Pro-angiogenic monocytes from PB-MNCs are considered as one of candidates for angiogenic therapy in regenerative medicine. Indeed, in a recent German clinical post-infarction remodeling study (TOPCARE-AMI) for ischemic heart disease, the ex vivo culture of PB-MNCs was employed. However, in this trial, there were different therapeutic efficacies in each case, possibly due to the different expansion efficacy of the ex vivo culture of PB-MNCs using autologous serum.In order to resolve this issue, we developed a new serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Floating spheres obtained by this serum-free culture system were mainly composed of CD11b+ monocytes. Interestingly, the mRNA expression of c-Mpl (thrombopoietin receptor) was markedly elevated compared with PB-MNCs, suggesting c-Mpl agonist could increase angiogenic property of cultured CD11b+ monocytes. Therefore, we assessed the impact of c-Mpl agonists on PB-MNC cultures in our serum-free method composed of X-VIVO15 medium with VEGF and bFGF.Both recombinant human thrombopoietin (rHuTPO) and romiplostim, a clinical grade second-generation TPO-receptor agonist, successfully increased sphere formations regarding both the number and size. The expressions of angiogenic factors, IL-8, CXCR4, and vasohibin-2, mRNA of CD11b+ monocytes cultured with c-Mpl agonists were up-regulated, indicating that cultivated CD11b+ monocytes have a proangiogenic potential.Finally, we investigated the proangiogenic potential of PB-MNCs derived CD11b+ monocytes in a hindlimb ischemia model utilizing BALB/c nude mice. Mice were randomly assigned to 7 groups: control mouse group (PBS-injected), freshly isolated CD11b+ monocyte-injected mouse group, cultivated CD11b+ monocyte with 2ng/ml and 20ng/ml rHuTPO -injected mouse groups, cultivated CD11b+ monocyte with 100ng/ml and 1000ng/ml romiplostim -injected mouse groups, cultivated CD11b+ monocyte without rHuTPO and romiplostim -injected mouse group. The intramuscular injection of CD11b+ monocytes cultivated with 20 ng/ml rHuTPO into the ischemic limb completely rescued the limbs from auto-amputations or foot necrosis, while only one (10.0%) of the control mice could be rescued. In addition, the intramuscular injection of both freshly isolated CD11b+ monocytes and CD11b+ monocytes cultivated without rHuTPO and romiplostim had a weak rescue effect on the ischemic limbs (8 and 7 of 10 mice had auto-amputations or foot necrosis, respectively). The salvage rate from necrosis in cultivated CD11b+ monocyte with romiplostim-injected mouse group is also superior to that in cultivated CD11b+ monocyte without rHuTPO-injected mouse group. Analysis of blood perfusion by a laser Doppler perfusion imaging system showed a significantly higher recovery in mice receiving the CD11b+ monocytes cultivated with 2 ng/ml or 20 ng/ml rHuTPO or 100ng/ml romiplostim 1 week after surgery. The functional capillary density and surface area visualized by perfusion with BS-I lectin also significantly increased in the rHuTPO- or romiplostim-treated group.In conclusion, an ex vivo addition of c-Mpl agonists augmented the pro-angiogenic activity of peripheral CD11b+ monocytes, and this method would be promising for human cell therapy to induce vascular regeneration by activating the angiogenic property in human peripheral blood-derived monocytes. Disclosures:Mizukami:The New Energy and Industrial Technology Development Organization of Japan: Research Funding.

Neutrophils Of Multiple Myeloma Are Dysfunctional and Immunosuppressive

IntroductionMultiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. However the role and function of neutrophils in MM and monoclonal gammopathy of undetermined significance (MGUS) has been poorly investigated. Methodson neutrophils (N) of 65 MM at diagnosis, 74 MGUS and 30 healthy subjects we evaluated, by flow cytometer, phagocytic activity and surface expression of CD64, CD16, CD62L and CD11b, markers of neutrophil activation. We tested also the immunosuppressive properties of N isolated from MGUS or MM patients, through functional assays, based on in vitro co-culture of N isolated from patients and T-lymphocytes from healthy subjects and we evaluated the expression of the immunosuppressive molecule arginase-1 (Arg-1) by RT-PCR. ResultsDespite no differences in the absolute number of N between MM, MGUS and healthy subjects, we found a functional impairment in MM not evident in MGUS patients.The phagocytic activity of MM-N was significantly reduced compared to healthy subjects-N (p<0.001) and MGUS-N (p<0.0001) and restored after induction chemotherapy (p=0.02).Expression of CD64 was significantly elevated in MM-N compared to MGUS-N or healthy subjects-N (p=0.01 and p= 0.007 respectively) and was inversely correlated with phagocytic activity (p=0,01). No differences were observed among MM, MGUS and healthy subjects for the other surface markers evaluated.MM-N exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. After PHA-P stimulation,T-lymphocytes isolated from healthy subjects missed the expression of activation markers such CD71, CD69, CD25, CD3ζ in the presence of MM-N for 72 hours, and in a less extensive way in the presence of MGUS-N. ConclusionCompared to controls, neutrophils obtained from MM patients have a reduced phagocytic activity, a greater expression of Arg-1 and exhibit an immunosuppressive function on T lymphocytes. Taken together, these findings indicated that neutrophils may contribute to impairment of immune function that characterizes MM patients. Disclosures:No relevant conflicts of interest to declare.

Parathyroid Hormone–Related Protein Drives a CD11b+Gr1+ Cell–Mediated Positive Feedback Loop to Support Prostate Cancer Growth

In the tumor microenvironment, CD11b(+)Gr1(+) bone marrow-derived cells are a predominant source of protumorigenic factors such as matrix metalloproteinases (MMP), but how distal tumors regulate these cells in the bone marrow is unclear. Here we addressed the hypothesis that the parathyroid hormone-related protein (PTHrP) potentiates CD11b(+)Gr1(+) cells in the bone marrow of prostate tumor hosts. In two xenograft models of prostate cancer, levels of tumor-derived PTHrP correlated with CD11b(+)Gr1(+) cell recruitment and microvessel density in the tumor tissue, with evidence for mediation of CD11b(+)Gr1(+) cell-derived MMP-9 but not tumor-derived VEGF-A. CD11b(+)Gr1(+) cells isolated from mice with PTHrP-overexpressing tumors exhibited relatively increased proangiogenic potential, suggesting that prostate tumor-derived PTHrP potentiates this activity of CD11b(+)Gr1(+) cells. Administration of neutralizing PTHrP monoclonal antibody reduced CD11b(+)Gr1(+) cells and MMP-9 in the tumors. Mechanistic investigations in vivo revealed that PTHrP elevated Y418 phosphorylation levels in Src family kinases in CD11b(+)Gr1(+) cells via osteoblast-derived interleukin-6 and VEGF-A, thereby upregulating MMP-9. Taken together, our results showed that prostate cancer-derived PTHrP acts in the bone marrow to potentiate CD11b(+)Gr1(+) cells, which are recruited to tumor tissue where they contribute to tumor angiogenesis and growth.

Multiple Lupus‐Associated ITGAM Variants Alter Mac‐1 Functions on Neutrophils

Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the nonsynonymous SNPs rs1143679, rs1143678, and rs1143683) are associated with systemic lupus erythematosus (SLE). ITGAM encodes the protein CD11b, a subunit of the β2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biologic functions of neutrophil Mac-1. Neutrophils from ITGAM-genotyped and -sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement-coated erythrocytes, serum-treated zymosan, heat-treated zymosan, and IgG-coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells, was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry. Mac-1-mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the β-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation. The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE.

Ex vivo activation of angiogenic property in human peripheral blood-derived monocytes by thrombopoietin

Human peripheral blood mononuclear cells (PB-MNCs) have angiogenic properties, which make them promising cells for use in angiogenic therapy approaches in regenerative medicine. To explore an efficient method for expanding pro-angiogenic cells from PB-MNCs, we developed a novel serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and thrombopoietin (TPO). Using this ex vivo culture, we obtained floating spheres composed mainly of CD11b(+) monocytes expressing c-Mpl (TPO receptor) and which exhibited acetylated low-density lipoprotein uptake and phagocytosis. Expression of IL-8, CXCR4, and vasohibin-2 mRNA was upregulated in these cells. In the presence of TPO, the number and size of the spheres were increased. In a nude mouse hind-limb ischemia model, the intramuscular injection of spheroid cells treated with TPO rescued blood perfusion more effectively than that without TPO. These results indicate that the ex vivo addition of TPO augments the pro-angiogenic activity of peripheral CD11b(+) monocytes, suggesting that this method shows promise for uses in human cell therapy aimed at the induction of vascular regeneration by activating the angiogenic properties of human peripheral blood-derived monocytes.

Immunobiotic Lactobacillus rhamnosus improves resistance of infant mice against respiratory syncytial virus infection

Previously we showed that orally administered Lactobacillus rhamnosus CRL1505 beneficially regulated the balance between pro- and anti-inflammatory mediators in the lungs of poly(I:C)-challenged mice, allowing an effective inflammatory response against the TLR3/RIG-I agonist but at the same time reducing tissue damage. The aim of the present study was to investigate whether oral administration of the CRL1505 strain was able to improve resistance against respiratory syncytial virus (RSV) infection in infant mice and to evaluate the immunological mechanisms involved in the immunobiotic effect. We demonstrated that treatment of 3-week old BALB/c mice with L. rhamnosus CRL1505 significantly reduce lung viral loads and tissue injuries after the challenge with RSV. Moreover, we showed that the protective effect achieved by the CRL1505 strain is related to its capacity to differentially modulate respiratory antiviral immune response. Our results shows that IFN-γ and IL-10 secreted in response to L. rhamnosus CRL1505 oral stimulation would modulate the pulmonary innate immune microenvironment conducting to the activation of CD103(+) and CD11b(high) dendritic cells and the generation of CD3(+)CD4(+)IFN-γ(+) Th1 cells with the consequent attenuation of the strong and damaging Th2 reactions associated with RSV challenge. Our results indicate that modulation of the common mucosal immune system by immunobiotics could favor protective immunity against respiratory viral pathogens with a high attack rate in early infancy, such as RSV.

Neutrophil activation by Campylobacter concisus

BackgroundCampylobacter concisus is an emerging enteric pathogen associated with prolonged diarrhoea and possibly inflammatory bowel disease in children as well as adults, but the interaction with cells of the innate immune system is unclear. The magnitude of systemic immunoglobulin response in acute infection is unknown.MethodsNeutrophils from healthy volunteers were activated with five faecal isolates of C. concisus from patients with gastroenteritis as well as the oral reference strain C. concisus ATCC33237. Neutrophils were tested for the expression of adherence molecule CD11b by immunoflourescence and for oxidative burst response by chemiluminescence. The opsonic activity in a chemiluminescence assay was assessed with heat treated serum from patients with C. concisus infection.ResultsA strong and dose-dependent activation of neutrophil adherence molecule CD11b and oxidative burst response was demonstrated with all six C. concisus isolates. Bacteria opsonised in heat treated serum induced an increased chemiluminescence response. Heat treated serum from patients with C. concisus infection did not have a higher opsonic activity than heat treated serum from healthy volunteers.ConclusionC. concisus has the capability to activate the innate immune system by stimulating neutrophil cells to increased adherence molecule expression and oxidative burst response, both crucial for acute inflammation. In a chemiluminescence assay the opsonic activity of heat treated serum from patients was not increased compared to heat treated control serum suggesting a weak systemic IgG response to infection.

A role for splenic monocytes/macrophages in acetaminophen-induced hepatotoxicity (P5140)

Abstract Activated macrophages (MP) has been implicated in hepatotoxicity induced by the analgesic acetaminophen (APAP). However, the origin of these cells is unknown. Here, we analyzed the role of splenic monocytes (Mo)/MP in APAP-induced liver inflammation and injury. Treatment of mice with APAP (300 mg/kg, i.p.) was associated with a time-related increase in CD11b+Ly6Chi pro-inflammatory MP in the liver after 24-96 h. In the spleen, but not the BM, this was correlated with a significant increase in the number and proliferative activity of CD11b+F4/80+Ly6Chi inflammatory Mo/MP. CD11b+F4/80-Ly6Chi inflammatory Mo/MP were also found to increase in the spleen, as well as in the BM 72-96 h post-APAP. Conversely, CD11b+F4/80+Ly6Clo anti-inflammatory Mo/MP decreased in the spleen after 48-96 h. To assess the role of splenic Mo/MP in APAP hepatotoxicity, we used splenectomized (spx) mice. APAP-induced hepatotoxicity was significantly reduced in spx mice, as measured by decreases in serum transaminases, and histological evidence of hepatic necrosis. This was associated with a decrease in pro-inflammatory Gal-3+ MP in the liver, and an increase in CX3CR1 and CCR2 expression, proteins important in recruiting anti-inflammatory MP into the liver following APAP-induced injury. Taken together, these results indicate that the spleen is a site of monocytopoiesis and that splenic Mo/MP contribute to inflammation and hepatotoxicity induced by APAP.

Complement Receptor 3 Influences Toll-like Receptor 7/8-Dependent Inflammation

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.

Activation of CD11b+ Kupffer Cells/Macrophages as a Common Cause for Exacerbation of TNF/Fas-Ligand-Dependent Hepatitis in Hypercholesterolemic Mice

We have reported that the mouse hepatic injury induced by either α-galactosylceramide (α-GalCer) or bacterial DNA motifs (CpG-ODN) is mediated by the TNF/NKT cell/Fas-ligand (FasL) pathway. In addition, F4/80+ Kupffer cells can be subclassified into CD68+ subset with a phagocytosing capacity and CD11b+ subset with a TNF-producing capacity. CD11b+ subset increase if mice are fed high-fat and cholesterol diet (HFCD). The present study examined how a HFCD affects the function of NKT cells and F4/80+ CD11b+ subset and these hepatitis models. After the C57BL/6 mice received a HFCD, high-cholesterol diet (HCD), high-fat diet (HFD) and control diet (CD) for four weeks, the HFCD mice increased surface CD1d and intracellular TLR-9 expression by the CD11b+ population compared to CD mice. Hepatic injury induced either by α-GalCer or CpG-ODN was more severe in HCD and HFCD mice compared to CD mice, which was in proportion to the serum TNF levels. In addition, liver cholesterol levels but not serum cholesterol levels nor liver triglyceride levels were involved in the aggravation of hepatitis. The FasL expression of NKT cells induced by both reagents was upregulated in HFCD mice. Furthermore, the liver mononuclear cells and purified F4/80+ CD11b+ subset from HFCD mice stimulated with either reagent in vitro produced a larger amount of TNF than did those from CD mice. Intracellular TNF production in F4/80+ CD11b+ cells was confirmed. The increased number of F4/80+ CD11b+ Kupffer cells/macrophages by HFCD and their enhanced TNF production thus play a pivotal role in TNF/NKT cell/FasL dependent hepatic injury.

Epidural anesthesia followed by epidural analgesia produces less inflammatory response than spinal anesthesia followed by intravenous morphine analgesia in patients with total knee arthroplasty

BackgroundAnesthesia and inflammatory response have been studied in major abdominal and thoracic surgical procedures, but not in major orthopaedic reconstructive procedures such as total knee arthroplasty. Most studies have compared general anesthesia with epidural anesthesia, but none has compared epidural with spinal.Material/MethodsIn a prospective randomized study, 2 groups of patients scheduled for total knee arthroplasty for osteoarthritis were evaluated regarding the inflammatory response to 2 types of regional anesthesia. In 30 patients (Group A) with spinal anesthesia followed by intravenous morphine analgesia, and in 26 patients (Group B) with epidural anesthesia followed by epidural analgesia, the inflammatory response was assessed through the calculation of leucocyte concentration (WBC), C-reactive protein (CRP), monocyte chemotactic protein 1 (MCP-1), interleukins (IL-1, IL-6, IL-10, IL-18), TNF-a, and leucocyte activation molecules CD11b and CD62l, in 3 blood samples (immediately before induction to anesthesia, immediately after closure of the operative wound, and at 24 hours post-operatively).ResultsThe MCP-1 values showed a statistically significant increase (p<0.02) in the group of patients with spinal anesthesia. Of the leucocyte activation molecules, a high statistically significant increase was noticed in the expression of CD11b on monocytes in the sample taken 24 hours post-operatively in the patients of group A. Similarly, CD62l expression on neutrophils showed a high statistically significant reduction in the sample taken 24 hours post-operatively in the group of patients with spinal anesthesia compared to the group of patients with epidural anesthesia.ConclusionsOur results show that epidural anesthesia followed by epidural analgesia produced less inflammatory response compared with spinal anesthesia followed by intravenous morphine analgesia in patients operated on with total knee arthroplasty, and that the most sensitive markers of those investigated were the CD11b and CD62l leucocyte activation molecules.

An expanded myeloid derived suppressor cell population does not play a role in gammaherpesvirus-exacerbated breast cancer metastases

BackgroundMice latently infected with murine gammaherpesvirus 68 (HV-68) and transplanted with 4 T1 breast cancer cells developed exacerbated metastatic lesions when compared to controls. The mechanisms responsible for this viral-exacerbated disease were not clear. The ability of HV-68 infection to induce S100A8 and S100A9 production and to expand a population of CD11b+Gr-1+ cells suggested that increased numbers, or activity, of viral-expanded myeloid derived suppressor cells (MDSCs) might contribute to HV-68-associated metastatic breast cancer in this model. We questioned whether mock or HV-68 infected mice with significant breast cancer might have differences in the number and/or activity of MDSCs.MethodsMyeloid-derived macrophages and dendritic cells were isolated from normal mice and cultured in vitro with HV-68 to assess S100A8 and S100A9 mRNA and protein expression. In vivo studies were performed using groups of mice that were mock treated or infected with HV-68. After viral latency was established, 4 T1 breast cancer cells were transplanted in mice. When primary breast tumors were present mice were euthanized and cells isolated for phenotyping of myeloid cell populations using FACS, and for ex vivo analysis of suppressor activity. Serum from these animals was also collected to quantify S100A8 and S100A9 levels.ResultsIn vitro studies demonstrated that direct exposure of myeloid cells to HV-68 did not induce increased expression of S100A8 or S100A9 mRNAs or secreted protein. HV-68 infected mice with metastatic breast cancer disease had no increases in S100A8/A9 levels and no significant increases in the numbers or activation of CD11b+Gr-1+MDSCs when compared to mock treated mice with breast cancer.ConclusionsTogether these studies are consistent with the notion that expanded myeloid derived suppressor cells do not play a role in gammaherpesvirus-exacerbated breast cancer metastases. The mechanisms responsible for HV-68 induced exacerbation of metastatic breast cancer remain unclear.

The rs1143679 (R77H) lupus associated variant of ITGAM (CD11b) impairs complement receptor 3 mediated functions in human monocytes

ObjectivesThe rs1143679 variant of ITGAM, encoding the R77H variant of CD11b (part of complement receptor 3; CR3), is among the strongest genetic susceptibility effects in human systemic lupus erythematosus (SLE)....

Long Lasting Local and Systemic Inflammation after Cerebral Hypoxic ischemia in Newborn Mice

BackgroundHypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice.Methodology/Principal FindingsWe developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b+ and CD11c+ antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb+ T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb− T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls.Conclusions/SignificanceOur findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks after damage, while the effects of the late inflammation phase may be missed. Furthermore, the self-reactive component raises the question if there is a correlation with development of autoimmune brain disease later in life.

Early Induction of CCL7 Downstream of TLR9 Signaling Promotes the Development of Robust Immunity to Cryptococcal Infection

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Response to Comment on “Adenosinergic Regulation of the Expansion and Immunosuppressive Activity of CD11b+Gr1+ Cells”

In our recent publication in The Journal of Immunology , we reported that the granulocytic CD11b+Gr1high subset of myeloid-derived suppressor cells (MDSCs) is significantly decreased in tumors growing in animals lacking A2B adenosine receptors, suggesting that this adenosine receptor subtype plays a

Comment on “Adenosinergic Regulation of the Expansion and Immunosuppressive Activity of CD11b+Gr1+ Cells”

In their recent article, Ryzhov et al. ([1][1]) reported that the granulocytic CD11b + Gr1high subset of myeloid-derived suppressor cells (MDSCs) generated from mouse hematopoietic progenitor cells in vitro displayed the highest levels of CD73 (ecto-5′-nucleotidase) expression followed by CD11b +