Maciej Siedlar

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)-Lys4-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.

Tumor cell-induced deactivation of human monocytes.

Although blood monocytes exhibit significant cytotoxic activity against tumor cells, the function of tumor infiltrating macrophages (TIM) is depressed in cancer patients. This study addresses the question of how the antitumor response of human monocytes, assessed by production of cytokines (tumor necrosis factor α, TNF; IL‐10; IL‐12p40) and cytotoxicity, is altered by exposure to cancer cells. Tumor cell−pre‐exposed monocytes restimulated with tumor cells showed significantly decreased production of TNF, IL‐12, increased IL‐10 (mRNA and release) and inhibition of IL‐1 receptor‐associated kinase‐1 (IRAK‐1) expression. This down‐regulation of cytokine production was selective, as the response of pre‐exposed monocytes to lipopolysaccharide (LPS) was unaffected. Treatment of tumor cell−pre‐exposed monocytes with hyaluronidase (HAase) improved their depressed production of TNF, while HAase‐treated cancer cells did not cause monocyte dysfunction. The response of hyaluronan (HA)−pre‐exposed monocytes to stimulation with tumor cells was also inhibited. Cytotoxic activity of monocytes pretreated with cancer cells was also decreased. This study shows that tumor cells selectively deactivate monocytes and suggests that tumor cell‐derived HA by blocking CD44 on monocytes inhibits their antitumor response. These observations may provide some explanation for the depressed function of TIM in human malignancy.

Tolerance Induced by the Lipopeptide Pam3Cys Is Due to Ablation of IL-1R-Associated Kinase-1

Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride (Pam3Cys) at 10 μg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam3Cys at 1 μg/ml leads to a reduced response after subsequent stimulation with Pam3Cys at 10 μg/ml, indicating that the cells have become tolerant to Pam3Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-κB binding in Pam3Cys-tolerant cells shows a failure to mobilize NF-κB-p50p65 heterodimers, while NF-κB-p50p50 homodimers remain unchanged. Pam3Cys-tolerant cells showed neither IκBα-Ser32 phosphorylation nor IκBα degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam3Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam3Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.

Isolation of extracellular vesicles: Determining the correct approach (Review)

The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this review was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this ‘uniqueness’ is the first step in the right direction for the complete assessment of EVs.

INTRAVESICAL INTERLEUKIN-2 IN T1 PAPILLARY BLADDER CARCINOMA: REGRESSION OF MARKER LESION IN 8 OF 10 PATIENTS

PURPOSE We evaluate the therapeutic effect of intravesical interleukin-2 (IL-2) on T1 papillary bladder carcinoma after incomplete transurethral resection. MATERIALS AND METHODS After incomplete transurethral resection we treated 10 patients in whom the marker lesion was left in place with 3 x 10(6) Chiron units IL-2 in 50 ml. saline plus 0.1% human serum albumin. The solution remained in the bladder for 2 hours and it was instilled on 5 consecutive days. The effect of IL-2 treatment on the marker lesion was evaluated by cystoscopy and repeat biopsy of the marker site 2 months after treatment. In addition, the effect on the recurrence of bladder tumors was studied. RESULTS At 2 months 8 of the 10 marker lesions (80%) had completely regressed and there were no tumor cells on repeat biopsy. Four patients remained tumor-free after 30 to 54 months. We noted no toxic effects. In 1 patient with a 7-year history of bladder cancer the marker was only partially regressed after 2 months. After removal of the marker this patient remained tumor-free at a followup of 54 months. CONCLUSIONS To our knowledge this report represents the first study of the effect of IL-2 on marker lesions left in place after transurethral resection. The results indicate that IL-2 instillations are feasible, and the combination of transurethral resection and IL-2 instillation may have a powerful antitumor effect. The therapeutic effects may not simply be due to intravesical IL-2, because previous transurethral resection probably caused some influx of infiltrating cells and the marker may have had tumor associated antigens. Consequently these effects may be due to the interaction of tumor associated antigens, infiltrating cells and IL-2.

Cross-talk between human monocytes and cancer cells during reactive oxygen intermediates generation: The essential role of hyaluronan

Human monocytes exhibit considerable cytocidal activity against tumor (but not normal cells) associated, at least partly, with the generation of reactive oxygen intermediates (ROIs). The present study examined the role of surface determinants and hyaluronan (HA) in the induction of ROI production by human monocytes stimulated with cancer cells, as measured by luminol‐enhanced chemiluminescence (CL). The inhibitory effect of monoclonal antibodies (MAbs) indicated the engagement of CD18, CD29 and CD44 adhesion molecules. Preincubation of monocytes and tumor cells, expressing CD44 determinants, with either anti‐CD44 MAb or HA inhibited CL generation. Addition of HA to monocytes decreased the expression of CD44 and induced CL response. Supernatants from the cultures of tumor cells stimulated CL response of monocytes, an effect that was abolished by treatment of the supernatants with hyaluronidase (HAase) or by preincubation of monocytes with an anti‐CD44 MAb. These results indicate that several surface molecules of monocytes, including CD44, are required to trigger the generation of ROI after their contact with tumor cells, whereas HA overexpressed on some cancer cells may allow monocytes (via CD44) to distinguish between transformed and normal cells. However, blocking of CD44 on monocytes by free HA dampens their response to tumor cells. Taken together, these observations suggest that the presence of HA in the tumor stroma may modulate effector functions of infiltrating macrophages and their interactions with cancer cells in situ.

Monoclonal antibodies against macrophage colony-stimulating factor diminish the number of circulating intermediate and nonclassical (CD14++CD16+/CD14+CD16++) monocytes in rheumatoid arthritis patient

To the editor: Human blood monocytes are divided into 3 subsets: classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++).[1][1] The latter 2 subpopulations produce inflammatory cytokines in response to a wide variety of pattern receptor ligands and are constantly

The Methods of Choice for Extracellular Vesicles (EVs) Characterization

In recent years, extracellular vesicles (EVs) have become a subject of intense study. These membrane-enclosed spherical structures are secreted by almost every cell type and are engaged in the transport of cellular content (cargo) from parental to target cells. The impact of EVs transfer has been observed in many vital cellular processes including cell-to-cell communication and immune response modulation; thus, a fast and precise characterization of EVs may be relevant for both scientific and diagnostic purposes. In this review, the most popular analytical techniques used in EVs studies are presented with the emphasis on exosomes and microvesicles characterization.

Functional links between Snail-1 and Cx43 account for the recruitment of Cx43-positive cells into the invasive front of prostate cancer

Suppressive function of connexin(Cx)43 in carcinogenesis was recently contested by reports that showed a multifaceted function of Cx43 in cancer progression. These studies did not attempt to model the dynamics of intratumoral heterogeneity involved in the metastatic cascade. An unorthodox look at the phenotypic heterogeneity of prostate cancer cells in vitro enabled us to identify links between Cx43 functions and Snail-1-regulated functional speciation of invasive cells. Incomplete Snail-1-dependent phenotypic shifts accounted for the formation of phenotypically stable subclones of AT-2 cells. These subclones showed diverse predilection for invasive behavior. High Snail-1 and Cx43 levels accompanied high motility and nanomechanical elasticity of the fibroblastoid AT-2_Fi2 subclone, which determined its considerable invasiveness. Transforming growth factor-β and ectopic Snail-1 overexpression induced invasiveness and Cx43 expression in epithelioid AT-2 subclones and DU-145 cells. Functional links between Snail-1 function and Cx43 expression were confirmed by Cx43 downregulation and phenotypic shifts in AT-2_Fi2, DU-145 and MAT-LyLu cells upon Snail-1 silencing. Corresponding morphological changes and Snail-1 downregulation were seen upon Cx43 silencing in AT-2_Fi2 cells. This indicates that feedback loops between both proteins regulate cell invasive behavior. We demonstrate that Cx43 may differentially predispose prostate cancer cells for invasion in a coupling-dependent and coupling-independent manner. When extrapolated to in vivo conditions, these data show the complexity of Cx43 functions during the metastatic cascade of prostate cancer. They may explain how Cx43 confers a selective advantage during cooperative invasion of clonally evolving, invasive prostate cancer cell subpopulations.

The B-cell Compartment in the Peripheral Blood of Children With Different Types of Primary Humoral Immunodeficiency

The aim of this study was to evaluate the B-cell compartment in the peripheral blood of children with different types of hipogammaglobulinemia: common variable immunodeficiency (CVID), transient hypogammaglobulinemia of infancy (THI), and selective IgA deficiency (SIgAD). We analyzed by flow cytometry the changes in the B-cell subsets with age and showed that children with an early-onset CVID develop similar pattern of B-cell subsets as adult patients with CVID with age, as the levels of memory B cells (CD19+/CD27+) and class-switched memory B cells (CD19+/CD27+/IgD−/IgM−), in contrast to age-matched control group, did not increase with age. Children with SIgAD displayed similar changes as patients with CVID only within the class-switched memory B-cell subpopulation. No significant differences in the level of memory B cells and class-switched memory B cells in children with THI in comparison to age-matched control group were observed. There were no differences in the percentage of immature B cells (CD19+/CD21low) among all studied groups. As B-cell subsets in children with THI were normal during entire period of hypogammaglobulinemia, the persistence of low levels of memory B-cell subsets in some children may facilitate the diagnosis of CVID.