Characterization of the CD14++CD16+ Monocyte Population in Human Bone Marrow

Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and nonclassical CD14⁺CD16⁺⁺ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14⁺⁺CD16⁺ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14⁺⁺CD16⁺ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14⁺⁺CD16⁻ and CD14⁺CD16⁺⁺ subsets which appear enriched in the periphery.     

Senescent CD14+CD16+ monocytes exhibit proinflammatory and proatherosclerotic activity

In elderly subjects and in patients with chronic inflammatory diseases, there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype, whose origin and functional relevance has not been well characterized. In this study, we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells, and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence, intracellular cytokine expression, ability to interact with endothelial cells, and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p < 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%, respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209, release of cytokines in response to low-intensity stimulus, and increased capacity to sustain lymphocyte proliferation. Finally, compared with CD14(++)CD16(-) cells, CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary, our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells, with increased inflammatory activity and ability to interact with endothelial cells. Therefore, accumulation of senescent monocytes may explain, in part, the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases.     

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6 ^-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6 ^-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6 ^-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4⁺ cells and a reduction in Th17 (CD4⁺IL17⁺) and mature dendritic (MHCII⁺CD11c⁺) cells recruitment. Clodronate depletion of macrophages in Ccr6 ^-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.     

A Higher Frequency of CD14+CD169+ Monocytes/Macrophages in Patients with Colorectal Cancer

Objective

Monocytes and macrophages can infiltrate into tumor microenvironment and regulate the progression of tumors. This study aimed at determining the frequency of different subsets of circulating monocytes and tumor infiltrating macrophages (TIMs) in patients with colorectal cancer (CRC).

Methods

The frequency of different subsets of circulating monocytes was characterized in 46 CRC patients and 22 healthy controls (HC) by flow cytometry. The frequency of different subsets of macrophages was analyzed in TIMs from 30 tumor tissues and in lamina propria mononuclear cells (LPMCs) from 12 non-tumor tissues. The concentrations of plasma cytokines and carcinoembryonic antigen (CEA) were determined. The potential association of these measures with the values of clinical parameters was analyzed.

Results

In comparison with that in the HC, the percentages of circulating CD14⁺CD169⁺, CD14⁺CD169⁺CD163⁺ and CD14⁺CD169⁺CD206⁺ monocytes and TIMs CD14⁺CD169⁺ as well as IL-10⁺CD14⁺CD169⁺, but not IL-12⁺ CD14⁺CD169⁺ macrophages were significantly increased, accompanied by higher levels of plasma IL-10 in the CRC patients. The percentages of CD14⁺CD169⁺ circulating monocytes and TIM macrophages were associated with the stage of disease and correlated positively with the levels of plasma IL-10 and CEA in CRC patients.

Conclusion

Our data suggest that an increase in the frequency of CD14⁺CD169⁺ cells may be associated with the development and progression of CRC and is concomitant rise of both, pro-tumor (M2-like, IL-10 producing) and anti-tumor (M1-like, IL-12 producing) monocytes and infiltrating macrophages. The frequency of CD14⁺CD169⁺ circulating monocytes and infiltrating macrophages may serve as a biomarker for evaluating the pathogenic degrees of CRC.     

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1a^high LCs form a dense network in the epidermis, the CD14⁺ and CD1a⁺ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a⁺ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14⁺ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a⁺ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.     

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma

Background

Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied.

Methods

We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry.

Results

Subjects with severe asthma had higher PAR-2 expression on CD14⁺⁺CD16⁺ monocytes (intermediate monocytes) and also higher percentage of CD14⁺⁺CD16⁺PAR-2⁺ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14⁺⁺CD16⁺PAR-2⁺ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14⁺⁺CD16⁺ PAR-2⁺ cells in peripheral blood compared to subjects without exacerbations.

Conclusions

PAR-2 expression is increased on CD14⁺⁺CD16⁺ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14⁺⁺CD16⁺ monocytes may play a role in the pathogenesis of severe asthma.     

Arachidonic Acid and Docosahexaenoic Acid Suppress Osteoclast Formation and Activity in Human CD14+ Monocytes,In vitro

An unbalanced diet can have adverse effects on health. Long chain polyunsaturated fatty acids (LCPUFAs) have been the focus of research owing to their necessity of inclusion in a healthy diet. However, the effects of LCPUFAs on human osteoclast formation and function have not been explored before. A human CD14+ monocyte differentiation model was used to elucidate the effects of an ω-3 LCPUFA, docosahexaenoic acid (DHA), and an ω-6 LCPUFA, arachidonic acid (AA), on osteoclast formation and activity. CD14+ monocytes were isolated from peripheral blood of healthy donors and stimulated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand to generate osteoclasts. Data from this study revealed that both the LCPUFAs decreased osteoclast formation potential of CD14+ monocytes in a dose-dependent manner when treated at an early stage of differentiation. Moreover, when exposed at a late stage of osteoclast differentiation AA and DHA impaired the bone resorptive potential of mature osteoclasts without affecting osteoclast numbers. AA and DHA abrogated vitronectin receptor expression in differentiating as well as mature osteoclasts. In contrast, the degree of inhibition for calcitonin receptor expression varied between the LCPUFAs with only AA causing inhibition during osteoclast differentiation. Furthermore, AA and DHA down regulated the expression of key osteoclast-specific genes in differentiating as well as mature osteoclasts. This study demonstrates for the first time that LCPUFAs can modulate osteoclast formation and function in a human primary osteoclast cell line.     

Circulating CD33+ large mononuclear cells contain three distinct populations with phenotype of putative antigen-presenting cells including myeloid dendritic cells and CD14+ monocytes with their CD16+ subset

BACKGROUND: In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. METHODS: To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. RESULTS: Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. CONCLUSIONS: This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC.     

The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF

In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.     

Dopamine Increases CD14+CD16+ Monocyte Migration and Adhesion in the Context of Substance Abuse and HIV Neuropathogenesis

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14⁺CD16⁺ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14⁺CD16⁺ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14⁺CD16⁺ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14⁺CD16⁺ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14⁺CD16⁺ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14⁺CD16⁺ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393, increased CD14⁺CD16⁺ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14⁺CD16⁺ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14⁺CD16⁺ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14⁺CD16⁺ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14⁺CD16⁺ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14⁺CD16⁺ monocytes in dopamine rich brain regions.     

Circulating CXCR5+CD4+ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1⁺ICOS⁺, PD1⁺ ICOS^-, or PD1^-ICOS^-. We used these markers to identify different subsets of CXCR5⁺CD4⁺ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD^-CD38^HiCD27⁺ memory B cells by flow cytometry. PD1⁺ICOS⁺ CXCR3⁺CCR6^-CXCR5⁺CD4⁺ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1⁺ICOS⁺ CXCR3^-CCR6^-CXCR5⁺CD4⁺ (Tfh2-like) cells for both vaccines, and PD1⁺ICOS⁺ CXCR3^-CCR6⁺CXCR5⁺CD4⁺ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1⁺ICOS⁺Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5⁺CD4⁺ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.     

WNT Signaling Pathway Gene Polymorphisms and Risk of Hepatic Fibrosis and Inflammation in HCV-Infected Patients

Background

Chronic hepatitis C infection is the leading cause of hepatocellular carcinoma (HCC), a highly lethal malignancy with rapidly increasing prevalence in the United States. Little is known about genetic variations and HCC risk. This study aimed to determine if genetic variation in Wnt signaling pathway genes are associated with advanced hepatic fibrosis and inflammation risk in a hepatitis C virus (HCV) infected population.

Methods

We performed a genetic association cross-sectional study evaluating single nucleotide polymorphisms (SNPs) in 58 candidate genes and risk of FibroSURE-Acti Test determined advanced fibrosis (F3/F4-F4 advanced cases vs. F0-F3 mild controls) and inflammation (A2/A3-A3 advanced cases vs. A0-A2 mild controls). We calculated odds ratios (ORs) and 95% confidence intervals (CIs) employing multivariate logistic regression. Haplotypes were inferred by the HAPLO.STAT program, interactions were evaluated using multifactor dimensionality reduction (MDR) analysis.

Results

Among 425 chronically HCV-infected male veterans, 155 (37%) had advanced fibrosis and 180 (42%) had advanced inflammation. Of 3016 SNPs evaluated, eight were significantly associated with fibrosis risk (e.g., SFRP2 rs11937424: OR = 2.19, 95% CI 1.48-3.23, P = 0.00004), and seven were significantly associated with inflammation risk (e.g., SFRP1 rs16890282: OR = 2.15, 95% CI 1.39-3.16, P = 0.0004). MDR analysis identified overweight/obese, SOST rs1405952, SFRP2 rs11937424, and FZD4 rs11234870 as the best interaction model for predicting risk of fibrosis; whereas race/ethnicity, FZD1 rs1346665, and TBX3 rs1520177 as the best interaction model for predicting risk of inflammation.

Conclusions

Polymorphisms in several genes involved in the Wnt signaling pathway were associated with hepatic fibrosis or inflammation risk in HCV-infected males. Additional studies in other multi-ethnic HCV cohorts are needed to validate our findings in males and to assess if similar associations exist in chronically HCV-infected females.     

The CD14+CD16+ Inflammatory Monocyte Subset Displays Increased Mitochondrial Activity and Effector Function During Acute Plasmodium vivax Malaria

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax–infected patients display significant increase in circulating monocytes, which were defined as CD14⁺CD16⁻ (classical), CD14⁺CD16⁺ (inflammatory), and CD14^loCD16⁺ (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16⁺ cells, in particular the CD14⁺CD16⁺ monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14⁺ were distinguished from CD14^lo monocytes by displaying larger and more active mitochondria. CD14⁺CD16⁺ monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14⁺CD16⁺ cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.     

Comparative Analysis of Hepatic CD14 Expression between Two Different Endotoxin Shock Model Mice: Relation between Hepatic Injury and CD14 Expression

CD14 is a glycoprotein that recognizes gram-negative bacterial lipopolysaccharide (LPS) and exists in both membrane-bound and soluble forms. Infectious and/or inflammatory diseases induce CD14 expression, which may be involved in the pathology of endotoxin shock. We previously found that the expression of CD14 protein differs among the endotoxin shock models used, although the reasons for these differences are unclear. We hypothesized that the differences in CD14 expression might be due to liver injury, because the hepatic tissue produces CD14 protein. We investigated CD14 expression in the plasma and liver in the carrageenan (CAR)-primed and D-galN-primed mouse models of endotoxin shock. Our results showed that severe liver injury was not induced in CAR-primed endotoxin shock model mice. In this CAR-primed model, the higher mRNA and protein expression of CD14 was observed in the liver, especially in the interlobular bile duct in contrast to D-galN-primed-endotoxin shock model mice. Our findings indicated that the molecular mechanism(s) underlying septic shock in CAR-primed and D-galN-primed endotoxin shock models are quite different. Because CD14 expression is correlated with clinical observations, the CAR-primed endotoxin shock model might be useful for studying the functions of CD14 during septic shock in vivo.     

Functions of Liver Natural Killer Cells Are Dependent on the Severity of Liver Inflammation and Fibrosis in Chronic Hepatitis C

During chronic hepatitis C virus (HCV) infection, the role of intra-hepatic (IH) natural killer (NK) cells is still controversial. To clarify their functions, we investigated anti-viral and cytotoxic activity of NK cells in human fresh liver biopsies. We compared the functions of IH-NK cells in HCV-infected and NASH patients in physiological conditions as well as after stimulation using flow cytometric and immunohistochemical analyses. Interestingly, few IH-NK cells produced anti-viral cytokine IFN-γ in HCV-infected patients similarly as in non-infected individuals. Spontaneous degranulation activity was extremely low in peripheral NK cells compared to IH-NK cells, and was significantly higher in IH-NK cells from HCV-infected patients compared to non-infected individuals. Immunohistochemical analysis revealed that perforin granules were polarized at the apical pole of IH-NK cells. The presence of CD107a and perforin in IH-NK cells demonstrated that NK cells exerted a cytolytic activity at the site of infection. Importantly, IH-NK cell functions from HCV-infected patients were inducible by specific exogenous stimulations. Upon ex vivo K562 target cell stimulations, the number of degranulating NK cells was significantly increased in the pool of IH-NK cells compared to circulating NK cells. Interestingly, after stimulation, the frequency of IFN-γ-producing IH-NK cells in HCV-infected patients was significantly higher at early stage of inflammation whereas the spontaneous IH-NK cell degranulation activity was significantly impaired in patients with highest inflammation and fibrosis Metavir scores. Our study highlights that some IH-NK cells in HCV-infected patients are able to produce INF-γ and degranulate and that those two activities depend on liver environment including the severity of liver injury. Thus, we conclude that critical roles of IH-NK cells have to be taken into account in the course of the liver pathogenesis associated to chronic HCV infection.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8⁺ T cells. However, their independent functional contributions in distinct CD8⁺ T cell subsets are not well understood. In this study, CD8⁺ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) CD8⁺ T cells (CD28^Hi), while its expression was significantly lower in effector (CD45RA⁺CD27⁻) CD8⁺ T cells (CD28^Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8⁺ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8⁺ T cells. Co-stimulation of CD28^Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28^Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28^Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8⁺ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8⁺ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8⁺ T cells. However, boosting a recall immune response via memory CD8⁺ T cells or vaccination to stimulate naïve CD8⁺ T cells would require CD28-mediated co-stimulation.     

CD4+ Cells Regulate Fibrosis and Lymphangiogenesis in Response to Lymphatic Fluid Stasis

Introduction

Lymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema.

Methods

We used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis.

Results

Lymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model.

Conclusions

Lymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis.