Juliusz Pryjma

Peripheral Blood CD14high CD16+ Monocytes are Main Producers of IL‐10

Based on CD14 and CD16 expression, human peripheral blood monocytes (MO) can be divided into a major CD14high CD16− population and two minor CD14high CD16+ and CD14dim CD16+ subpopulations. CD14dim CD16+ MO are well characterized and regarded as pro‐inflammatory because upon stimulation produce TNF‐α but little, if any, IL‐10. By contrast, little is known about CD14high CD16+ MO. We investigated the surface expression of selected determinants by CD16+ MO subpopulations, cytokine production, phagocytosis and antigen presentation. We found that both CD16+ subpopulations had a higher expression of HLA‐DR, CD86, CD54 and a lower expression of CD64 than CD14high CD16− population. In addition, CD14high CD16+ MO showed a higher expression of CD11b and TLR4 than CD14dim CD16+ and CD14high CD16− subpopulations. CD14high CD16+ MO exhibited an increased phagocytic activity and a decreased antigen presentation in comparison with CD14dim CD16+. As expected, lipopolysaccharide (LPS)‐stimulated CD14dim CD16+ MO produced TNF‐α but little IL‐10. By contrast, LPS‐stimulated CD14high CD16+ subpopulation produced significantly more IL‐10 than CD14dim CD16+ and CD14high CD16− MO. In conclusion, our data show that human peripheral blood CD16+ MO are heterogeneous in function and consist of two subpopulations: CD14dim CD16+ pro‐inflammatory and CD14high CD16+ with anti‐inflammatory potential.

Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete "sub-G1" peaks on DNA content histograms.

Internucleosomal DNA fragmentation is one of the hallmarks of apoptosis. Because the low molecular weight DNA fragments are extracted during cell staining in aqueous solutions, apoptotic cells can be identified on DNA content frequency histograms as cells with fractional (“sub‐G1”) DNA content. The aim of the present study was to explore whether in situ DNA fragmentation during apoptosis is discontinuous or progresses incessantly and if it is discontinuous, to define the resistant to cleavage fraction of DNA that remains stainable with the fluorochrome.

CD14+CD16+ monocytes in the course of sepsis in neonates and small children: Monitoring and functional studies

The phenotype and function of peripheral blood monocytes change after trauma and during sepsis. The aim of the study was to evaluate monocyte expression of human leucocyte antigen (HLA)‐DR and Fc receptor III (FcR III) (CD16) in neonates and small children with high risk of sepsis (hospitalized at the intensive care unit). The reduced proportion of CD14+HLA‐DR+ monocytes was observed in all patients at the intensive care unit, while the increase of CD16 expression on monocytes was observed in the course of sepsis. The measurement of CD16 expression on monocytes also proved to be more useful for monitoring patient. The proportion of both CD14dimCD16+ and CD14highCD16+ monocytes increased during sepsis; however, monocytes showed reduced ability to phagocytose Escherichia coli, compromised ability to cooperate with T cells and reduced CD86 expression in parallel to HLA‐DR depression. The reduced interleukin (IL)‐1 but rather increased IL‐10 production was associated with sepsis. The differences between CD14+CD16+ monocytes of healthy donors and patients with sepsis are discussed.

Persistent skin colonization with Staphylococcus aureus in atopic dermatitis: relationship to clinical and immunological parameters

Background Staphylococcus aureus has important implications for the pathogenesis of atopic dermatitis (AD). In some patients S. aureus can be eradicated from the skin during anti‐inflammatory treatment, while in others bacterial colonization is persistent. Potential mechanisms and features of these two distinct groups of patients are not known.

Serum cytochrome c indicates in vivo apoptosis and can serve as a prognostic marker during cancer therapy.

Despite significant progress in cancer therapy, the outcome of the treatment is often unfavorable. Better treatment monitoring would not only allow an individual more effective, patient‐adjusted therapy, but also it would eliminate some of the side effects. Using a cytochrome c ELISA that was modified to increase sensitivity, we demonstrate that serum cytochrome c is a sensitive apoptotic marker in vivo reflecting therapy‐induced cell death burden. Furthermore, increased serum cytochrome c level is a negative prognostic marker. Cancer patients whose serum cytochrome c level was normal 3 years ago have a twice as high probability to be still alive, as judged from sera samples collected for 3 years, analyzed recently and matched with survival data. Moreover, we show that serum cytochrome c and serum LDH‐activity reflect different stages and different forms of cell death. Cellular cytochrome c release is specific for apoptosis, whereas increased LDH activity is an indicator of (secondary) necrosis. Whereas serum LDH activity reflects the “global” degree of cell death over a period of time, the sensitive cytochrome c‐based method allows confirmation of the individual cancer therapy‐induced and spontaneous cell death events. The combination of cytochrome c with tissue‐specific markers may provide the foundation for precise monitoring of apoptosis in vivo, by “lab‐on‐the‐chip” technology.

Fas (CD95)-Fas Ligand Interactions Are Responsible for Monocyte Apoptosis Occurring as a Result of Phagocytosis and Killing of Staphylococcus aureus

ABSTRACT Human peripheral blood monocytes become apoptotic following phagocytosis of Staphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis of S. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 preventsS. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis of S. aureus involves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.

Human MO subsets as defined by expression of CD64 and CD16 differ in phagocytic activity and generation of oxygen intermediates.

Phagocytosis and killing of microorganisms by reactive oxygen radicals are important defence mechanisms of the immune system and it was shown that human monocytes (MO) are heterogeneous in exerting these functions. Previously, we described that human peripheral blood MO consist of a major subset of Fc gamma-receptor-I (CD64)-positive cells exhibiting low accessory cell capacity but high phagocytic activity, and a minor subset of CD64-negative cells with dendritic cell (DC)-like high T cell accessory cell capacity but low phagocytic capacity. Recently, we could show that each subset itself further differs in the expression of the Fc gamma-receptor-III (CD16) and T cell accessory activities resulting in four different subsets: two CD16+ subsets (CD64+ or CD64-) with high T cell stimulation capacity and two CD16- subsets (CD64+ or CD64-) with low accessory activities. In the present study we demonstrate that these subsets also differ in their ability to phagocytose opsonized bacteria (S. aureus and E. coli) and in the generation of reactive oxygen species. Both CD64+ subsets (CD16+ or CD16-) exhibit high phagocytic activity accompanied by intracellular superoxide induction. Luminol-dependent (mainly myeloperoxidase (MPO)-mediated) chemiluminescence (CL) response to latex and FMLP (formylmethionylleucylphenylalanine) was also high in these cell populations. Phagocytic activity and modest CL response was shown in CD64-/CD16+ but not in CD64-/CD16- cells, indicating that each subset except for CD64-/CD16- cells may engulf bacteria and exhibit MPO activity. Taken together, these data demonstrate further heterogeneity of peripheral blood MO in both, phagocytic activity and generation of reactive oxygen species indicating differences between the four subsets in this kind of defence mechanisms against pathogens.

The Kinase Activity of IL-1 Receptor-associated Kinase 4 Is Required for Interleukin-1 Receptor/Toll-like Receptor-induced TAK1-dependent NFκB Activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFκB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKα/β phosphorylation and IKKβ activation, resulting in classic NFκB activation through IκBα phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKγ phosphorylation and IKKα activation, resulting in NFκB activation through dissociation of phosphorylated IκBα from NFκB without IκBα degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFκB activation pathway, but mediated IL-1-induced TAK1-independent NFκB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFκB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFκB activation.

The role of cytokines in monocyte apoptosis.

Survival or apoptosis, activation and differentiation, phagocytosis and antigen presentation, migration or participation in granuloma formation are features of freshly recruited blood-borne monocytes in the local environment. In this presentation we describe that human monocytes undergo spontaneous apoptosis in vitro which involves Fas/FasL interactions, and that proinflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β and granulocyte-monocyte-colony-stimulating factor prevent spontaneous apoptosis. In vitro infection of purified monocytes with low numbers of Mycobacterium tuberculosis H37Rv prevents spontaneous apoptosis. The apoptosis-preventing effect is correlated to the release of TNFα and not due to phagocytosis per se. Furthermore, the minor subset of CD64-negative monocytes is found to be less susceptible to recall antigen-activated CD4-positive T cell-mediated apoptosis than CD64-positive monocytes. Finally, recent findings of our group indicate that the chemokine platelet factor 4 protects monocytes from spontaneous apoptosis and induces the differentiation of monocytes into macrophages. From these findings we conclude that monocyte recruitment, their survival, their differentiation and their functional activity at the site of inflammation are regulated by a cytokine network which needs to be further analyzed in order to design strategies for immune intervention.

Increased IL-10 production during spontaneous apoptosis of monocytes.

Monocytes/macrophages undergo apoptosis and are in contact with apoptotic cells both in vitro and in vivo. The data show that monocytes undergoing spontaneous apoptosis in vitro change their cytokine production profile. We demonstrate that the lipopolysaccharide (LPS)‐induced production of interleukin‐10 (IL‐10) is up‐regulated, while production of pro‐inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) is either not affected or reduced. These differences seen both at the protein and mRNA level directly correlate with the appearance of apoptotic cells in the culture. Flow cytometry analysis using double staining, surface with annexin V and intracellular with anti‐IL‐10, suggested that annexin V‐negative monocytes are the predominant source of IL‐10. Analysis of sorted populations of monocytes indicated that the increase in IL‐10 synthesis appears to result from direct interactions between non‐apoptotic and apoptotic cells at the time of stimulation. Also non‐apoptotic, freshly isolated monocytes produced more IL‐10 upon stimulation with LPS, Staphylococcus aureus or zymosan when apoptotic neutrophils were added to the culture. In contrast, monocyte‐derived macrophages did not produce more IL‐10 in the presence of apoptotic neutrophils. Finally, we found that the presence of apoptotic monocytes in the culture may influence specific immune responses. The data show that in the presence of annexin V‐positive monocytes CD4‐positive memory T cells produce less IFN‐γ upon stimulation with purified protein derivative of tuberculin, which could be partially reversed by anti‐IL‐10 neutralizing antibodies. We conclude that these findings might illustrate the mechanisms operating within an inflammatory site and play an important immunoregulatory role during the resolution of inflammation and specific immune responses.