Interactions between innate and adaptive immune functions in neonatal macrophages (MΦ) are still unclear. We therefore established a method to quantify bacterial phagocytosis and intracellular degradation, using green fluorescent protein (GFP)‐labeled Escherichia coli in combination with phenotypic analysis. The kinetics of the proportion of phagocyting MΦ, phagocytosed bacteria per MΦ, and bacterial degradation were comparable for cord blood MΦ of term neonates and MΦ of healthy adults. Phenotyping revealed CD14 and CD16 to be down‐modulated within minutes. GFP‐labeled E. coli may be useful tools to further study MΦ subpopulations and determinants of phagocytosis in cord blood MΦ. © 2006 International Society for Analytical Cytology

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Background:The propensity for sustained inflammation after bacterial infection in neonates, resulting in inflammatory sequelae such as bronchopulmonary dysplasia and periventricular leucomalacia, is well known, but its molecular mechanisms remain elusive. Termination of inflammatory reactions physiologically occurs early after removal of bacteria by phagocytosis-induced cell death (PIcD) of immune effector cells such as monocytes. PIcD from cord blood monocytes (cBMOs) was shown to be reduced as compared with that of peripheral blood monocytes (PBMOs) from adult donors in vitro. Methods: PBMOs, cBMOs, and Fas (cD95)-deficient (lpr) mouse monocytes were analyzed in an in vitro infection model using green fluorescence protein-labeled Escherichia coli (E. coli-GFP). Phagocytosis and apoptosis were quantified by flow cytometry and cD95L secretion was quantified by enzyme-linked immunosorbent assay. results: We demonstrate the involvement of the cD95/ cD95 ligand pathway (cD95/cD95L) in PIcD and provide evidence that diminished cD95L secretion by cBMOs may result in prolonged activation of neonatal immune effector cells. conclusion: These in vitro results offer for the first time a molecular mechanism accounting for sustained inflammation seen in neonates.

Hematopoietic stem and progenitor cells (HSPCs) are known to reside in specialized niches at the endosteum in the trabecular bone. Osteoblasts are the major cell type of the endosteal niche. It is well established that secreted proteases are involved in cytokine-induced mobilization processes that release stem cell from their niches. However, migratory processes such as the regular trafficking of HSPCs between their niches and the periphery are not fully understood. In the current study we analyzed whether osteoblast-secreted cysteine cathepsins are able to reduce the direct interaction of HSPCs with bone-forming osteoblasts. Isolated human osteoblasts were shown to secrete proteolytically active cysteine cathepsins, such as cathepsins B, K, L, and X. All of these cathepsins were able to digest, although with different efficacy, the chemokine CXCL12, which is known to be important for retaining HSPCs in their niches. Of the 4 identified cathepsins, only cathepsin X was able to reduce binding of HSPCs to osteoblasts. Interestingly, nonactivated pro-cathepsin X and mature cathepsin X did not interfere with HSPC-osteoblast interactions. Only pro-cathepsin X treated with dithiothreitol, which unfolds but does not lead to full maturation of cathepsin X, significantly reduced HSPC adhesion to osteoblasts. These observations argue for a role of the accessible cathepsin X prodomain in diminishing cell binding. Our findings strongly suggest that the cysteine cathepsins B, K, and L constitutively secreted by osteoblasts are part of the fine-tuned regulation of CXCL12 in the bone marrow, whereas pro-cathepsin X with its prodomain can affect HSPC trafficking in the niche.

Septic diseases are characterized by an initial systemic, proinflammatory phase, followed by a period of anti-inflammation. In the context of the latter, monocytes have been described to display altered functions, including reduced TNF secretion and T cell-stimulating capacities in response to recall antigens. This hyporesponsiveness is supposed to be detrimental for coping with secondary infections. We here characterize bacterially reprogrammed PBMC-derived monocytes with special focus on their phagocytic activity. Hence, we have implemented a surrogate model of the early, postinflammatory period by exposing PBMCs to Escherichia coli on d0 and rechallenging them with bacteria on d2. This induced the emergence of a distinct monocytic phenotype with profound phagocytic impairments but a preserved ability for naïve T cell stimulation. The compromising effects on phagocytosis required the presence of bacteria and were not mimicked by TLR4 ligation or exposure to isolated cytokines alone. Moreover, the impairments were specific for the engulfment of bacteria and were coupled to a selective down-regulation of Fc␥R and SR expression. Intriguingly, this monocytic phenotype contributed to the stimulation of a T H 17-polarized adaptive immune response in the context of secondary infection. Our findings extend the current knowledge of monocytic reprogramming and identify the phagocytic capacity of monocytes as a putative sepsis biomarker. J. Leukoc. Biol. 91: 977-989; 2012. 2. These authors share senior authorship.Abbreviations: BAI1ϭbrain-specific angiogenesis inhibitor-1, CRϭ complement receptor, dϭday, IL-1RaϭIL-1R antagonist, ITGϭintegrin, Lox-1ϭlectin-like oxidized LDLR 1, MFIϭmean fluorescence intensity, oxLDLϭ oxidized LDL, PSϭphosphatidylserine, qϭquantitative, SRϭscavenger receptor, TIMϭT cell Ig and mucin domain-containing proteinThe online version of this paper, found at www.jleukbio.org, includes supplemental information.Article 0741-5400/12/0091-977