Prognostic and pathogenic role of CXC motif ligand 16 in sepsis

Chemokine CXC motif ligand 16 (CXCL16) is an important mediator that has been shown to participate in various human diseases. The role of CXCL16 in the immunopathology of sepsis remains unidentified. In this study, we found that human patients with sepsis had significantly higher soluble levels of serum CXCL16 than healthy volunteers on day of intensive care unit (ICU) admission. Soluble CXCL16 remained significantly up-regulated in the patients with sepsis, which correlated with disease severity. Furthermore, nonsurvivors displayed significantly higher admission levels of soluble CXCL16 compared with survivors of septic patients. Soluble CXCL16 levels revealed significant prognostic value for 28-day mortality, and CXCL16 was shown to be an independent predictor of 28-day mortality in the patients with sepsis. In a murine model of cecal ligation and puncture (CLP)-induced nonsevere sepsis, supplementation of recombinant CXCL16 protein could increase sepsis-induced mortality and tissue injury. Conversely, neutralizing CXCL16 by anti-CXCL16 monoclonal antibody could decrease mortality and tissue injury in CLP-induced severe sepsis. However, CXCL16 did not affect the ability of these mice to clear bacteria in CLP. Taken together, CXCL16 could be related to sepsis not only as a novel biomarker of prognosis, but also as a potential target for therapeutic intervention.     

The role of B cell CD22 expression in Staphylococcus aureus arthritis and sepsis

Severe Staphylococcus aureus infections give rise to a pronounced antigen-specific and polyclonal B cell response with elevated serum immunoglobulin levels. However, it has been difficult to correlate the antibody levels with the clinical outcome of sepsis and/or arthritis concerning both protection and pathogenic aspects. Earlier studies have shown that macrophages and neutrophils are of great importance for bacterial clearance. However, deletion of the complete B cell compartment affected neither S. aureus-induced arthritis nor survival. MZ B cells are believed to be of importance for clearance of blood-borne antigens and have been implicated in protection against S. aureus infections. CD22 is a B-cell-specific inhibitory receptor binding to alpha2,6-linked sialic acids, and deficiency in CD22 leads to a 75% reduction of the MZ B cell compartment. CD22-/- mice and congeneic controls were inoculated intravenously with an arthritogenic dose of live S. aureus. No differences between the groups were detected regarding frequency and severity of arthritis, survival, bacterial clearance, or induction of inflammatory response. This study shows explicitly that a reduced MZ B cell compartment in the absence of CD22 expression does not interfere with the inflammatory response during S. aureus infection.     

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.     

CD16 promotes Escherichia coli sepsis through an FcR gamma inhibitory pathway that prevents phagocytosis and facilitates inflammation

Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here we show that the FcRgamma adaptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing signal transduction subunit of the Fc receptor family, has a deleterious effect on sepsis. FcRgamma(-/-) mice show increased survival during peritonitis, owing to markedly increased E. coli phagocytosis and killing and to lower production of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The FcRgamma-associated receptor that inhibits E. coli phagocytosis is FcgammaRIII (also called CD16), and its absence protects mice from sepsis. FcgammaRIII binds E. coli, and this interaction induces FcRgamma phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibits E. coli phagocytosis and increases TNF-alpha production through Toll-like receptor 4. We identified the phagocytic receptor negatively regulated by FcRgamma on macrophages as the class A scavenger receptor MARCO. E. coli-FcgammaRIII interaction induces the recruitment of SHP-1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding FcgammaRIII, E. coli triggers an inhibitory FcRgamma pathway that both impairs MARCO-mediated bacterial clearance and activates TNF-alpha secretion.     

Expression of CD16, CD18 and CD56 in tributyltin-exposed human natural killer cells

Tributyltin (TBT) was produced in large quantities for use in wood preservation, marine antifouling paints, disinfection of circulating industrial cooling waters and slime control in paper mills. TBT is found in dairy products, meat and fish. We and others have shown that there are measurable levels of TBT in human blood. BTs appear to increase the risk of cancer and viral infections in exposed individuals. In previous studies, we demonstrated that the NK-cytotoxic function of lymphocytes was greatly diminished after a 1-h exposure to 300 nM TBT or a 24-h exposure to 200 nM TBT. Inhibition induced by a 1-h exposure to 300 nM TBT continues even after removal of the compound. There is also decreased ability of NK cells to bind to tumor target cells when they have been exposed to 200 nM TBT for 24 h. This loss of binding function is not seen when NK cells are exposed to 300 nM TBT for 1 h. However, NK cells exposed to 300 nM TBT for 1 h and then incubated in TBT-free media for 24, 48 or 96 h, show a significant loss of tumor-binding function by 96 h. The effects of TBT on cell surface molecules that are crucial to NK cell function is investigated. The data indicate there is a loss of expression of CD16 and CD56 on NK cells exposed to 200 nM TBT for 24 h. There is no decrease in expression of any of the markers studied when NK cells are exposed to 300 nM TBT for 1 h, consistent with the fact that a 1-h exposure has no effect on the ability of NK cells to bind to tumor targets. However, when NK cells are exposed to 300 nM TBT followed by 24, 48 or 96 h incubations in TBT-free media, there is a significant loss of CD16 and CD18 expression after 24 h and of CD16 and CD56 expression after 48 and 96 h.     

Deleterious Passengers in Adapting Populations

Most new mutations are deleterious and are eventually eliminated by natural selection. But in an adapting population, the rapid amplification of beneficial mutations can hinder the removal of deleterious variants in nearby regions of the genome, altering the patterns of sequence evolution. Here, we analyze the interactions between beneficial “driver” mutations and linked deleterious “passengers” during the course of adaptation. We derive analytical expressions for the substitution rate of a deleterious mutation as a function of its fitness cost, as well as the reduction in the beneficial substitution rate due to the genetic load of the passengers. We find that the fate of each deleterious mutation varies dramatically with the rate and spectrum of beneficial mutations and the deleterious substitution rate depends nonmonotonically on the population size and the rate of adaptation. By quantifying this dependence, our results allow us to estimate which deleterious mutations will be likely to fix and how many of these mutations must arise before the progress of adaptation is significantly reduced.     

Mesenchymal stromal cells impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activate monocytes

Background aimsMesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulationMethodsTo understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approachesResultsMSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell–cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytesConclusionsOur results suggest that MSC impair the differentiation of CD14⁺⁺ CD16^? CD64⁺ classical monocytes into CD14⁺⁺ CD16⁺ CD64⁺⁺ activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.     

Bacterial virulence plays a crucial role in MRSA sepsis

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.     

The role of CD95 and CD95 ligand in cancer

CD95 (Fas/APO-1) and its ligand, CD95L, have long been viewed as a death receptor/death ligand system that mediates apoptosis induction to maintain immune homeostasis. In addition, these molecules are important in the immune elimination of virus-infected cells and cancer cells. CD95L was, therefore, considered to be useful for cancer therapy. However, major side effects have precluded its systemic use. During the last 10 years, it has been recognized that CD95 and CD95L have multiple cancer-relevant nonapoptotic and tumor-promoting activities. CD95 and CD95L were discovered to be critical survival factors for cancer cells, and were found to protect and promote cancer stem cells. We now discuss five different ways in which inhibiting or eliminating CD95L, rather than augmenting, may be beneficial for cancer therapy alone or in combination with standard chemotherapy or immune therapy.     

Essential role of CD8 palmitoylation in CD8 coreceptor function

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.     

Selective mobilization of CD14(+)CD16(+) monocytes by exercise

Strenuous, anaerobic exercise leads to an increaseof leukocytes that are mobilized from the marginal pool. We haveanalyzed in human peripheral blood the effect of exercise on the number of CD14⁺CD16⁺ monocytes as determined bytwo-color immunofluorescence and flow cytometry. We show herein thatthis type of monocyte responds with a dramatic up to 4.8-fold increase.Mobilization does not occur after 1 min at 100 or 200 W but 1 min at400 W leads to a twofold increase of theCD14⁺CD16⁺ monocytes immediately afterexercise. The numbers remain high at 5 min and gradually decrease toreach the initial level at 20 min postexercise. After 20 min of rest,the CD14⁺CD16⁺ monocytes can be mobilized againby a second exercise. The CD14⁺CD16⁺ monocytesappear to be mobilized from the marginal pool where they preferentiallyhome because of a higher expression of adhesion molecules like CD11dand very late antigen-4. Exercise goes along with an increaseof catecholamines, and mobilization of theCD14⁺CD16⁺ monocytes can be substantiallyreduced by treatment of donors with the -adrenergic receptor blockerpropranolol. Mobilization of CD14⁺CD16⁺monocytes by a catecholamine-dependent mechanism may contribute to theincrease of these cells in various clinical conditions.

     

Identification of CD16-2, a novel mouse receptor homologous to CD16/Fc gamma RIII

It is believed that mouse Fc gamma RIII arose by an evolutionarily recent recombination, which brought together the extracellular domains from Fc gamma RII with the transmembrane/cytoplasmic region from the ancestor Fc gamma RIII. Here, we report identification of a mouse gene encoding a transmembrane receptor that may be regarded as the true ortholog of nonrodent CD16/Fc gamma RIII. Designated CD16-2, the novel protein is highly similar to human Fc gamma RIIIA in the signal peptide (60% identical residues), and in the extracellular domains (65%). Although the similarity between the two proteins is less conspicuous in the transmembrane/cytoplasmic region (54%), it is higher than between human Fc gamma RIIIA and mouse Fc gamma RIII (44%). However, the conserved transmembrane motif LFAVDTGL shared by rodent and human Fc gamma RIII and Fc epsilon RI has two replacements in CD16-2. The CD16-2 gene is tightly linked to the Fc gamma RIII and Fc gamma RII genes and consists of five exons. Northern blot analysis revealed that CD16-2 is expressed in peripheral blood leukocytes, as well as in spleen, thymus, colon and intestine. RT-PCR showed prominent expression in macrophage cell line J774. Based on sequence comparisons, it is suggested that the modern repertoire of the mammalian low affinity Fc receptors has resulted from repetitive duplications and/or recombinations of three ancestral genes.     

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14(hi) /CD16(neg) , CD14(hi) /CD16(pos) and CD14(lo) /CD16(pos) ) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi) /CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo) /CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi) /CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies. ? 2012 International Society for Advancement of Cytometry.     

Deleterious iron-mediated oxidation of biomolecules

Iron is an essential metal for most biological organisms. However, if not tightly controlled, iron can mediate the deleterious oxidation of biomolecules. This review focuses on the current understanding of the role of iron in the deleterious oxidation of various biomolecules, including DNA, protein, lipid, and small molecules, e.g., ascorbate and biogenic amines. The effect of chelation on the reactivity of iron is also addressed, in addition to iron-associated toxicities. The roles of the iron storage protein ferritin as both a source of iron for iron-mediated oxidations and as a mechanism to safely store iron in cells is also addressed.     

Deleterious GRM1 mutations in schizophrenia

We analysed a phenotypically well-characterised sample of 450 schziophrenia patients and 605 controls for rare non-synonymous single nucleotide polymorphisms (nsSNPs) in the GRM1 gene, their functional effects and family segregation. GRM1 encodes the metabotropic glutamate receptor 1 (mGluR1), whose documented role as a modulator of neuronal signalling and synaptic plasticity makes it a plausible schizophrenia candidate. In a recent study, this gene was shown to harbour a cluster of deleterious nsSNPs within a functionally important domain of the receptor, in patients with schizophrenia and bipolar disorder. Our Sanger sequencing of the GRM1 coding regions detected equal numbers of nsSNPs in cases and controls, however the two groups differed in terms of the potential effects of the variants on receptor function: 6/6 case-specific and only 1/6 control-specific nsSNPs were predicted to be deleterious. Our in-vitro experimental follow-up of the case-specific mutants showed that 4/6 led to significantly reduced inositol phosphate production, indicating impaired function of the major mGluR1 signalling pathway; 1/6 had reduced cell membrane expression; inconclusive results were obtained in 1/6. Family segregation analysis indicated that these deleterious nsSNPs were inherited. Interestingly, four of the families were affected by multiple neuropsychiatric conditions, not limited to schizophrenia, and the mutations were detected in relatives with schizophrenia, depression and anxiety, drug and alcohol dependence, and epilepsy. Our findings suggest a possible mGluR1 contribution to diverse psychiatric conditions, supporting the modulatory role of the receptor in such conditions as proposed previously on the basis of in vitro experiments and animal studies.