Moncef Guenounou

Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro

Calcium phosphate bioceramics have been applied as bone substitutes for several decades. Aseptic loosening after total joint arthroplasty is a major problem in orthopaedic surgery. Hydroxyapatite particles from materials wear have been reported as the main cause of implant failure. For this reason, an investigation into possible wear particles from materials used in the implant may lead to longevity after arthroplasty. Monocytes are among the first cells to colonize the inflammatory site. In the present study, we have evaluated the inflammatory response after exposition to particles with different characteristics (size, sintering temperature and shape). Our data demonstrate that the most important characteristic was the shape and the size of the particles. The needle shaped particles induced the larger production of TNF-alpha, IL-6 and IL-10 by cells. To a less manner, the smallest particles induced an increase of the expression and production of the cytokines studied (TNF-alpha, IL-6 and IL-10). The sintering temperature appeared to be a less important characteristic even though it was involved in the dissolution/precipitation process.

Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.

According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.

Effect of hydroxyapatite sintering temperature on intracellular ionic concentrations of monocytes: A TEM‐cryo‐x‐ray microanalysis study

Hydroxyapatite used as bone replacement can lead to particle release in the implantation site. These particles interact with monocytes, which are the first immune cells to colonize the implant and an inflammatory site. Thanks to cryo-X-ray microanalysis, we can observe cells in a state close to the physiological one and we have access to diffusible ions. We paid particular attention to the potassium-to-sodium ratio, which is one of the best viability criteria. We used this method to study the interaction between three hydroxyapatite particles treated at three different temperatures (not treated, treated at 600 degrees C and 1180 degrees C), and monocytes. In the culture condition, the hydroxyapatite treated at 1180 degrees C underwent the least dissolution. We demonstrate that monocytes were altered by the three hydroxyapatite particles. The hydroxyapatite particules treated at 600 degrees C were found to be more toxic.

Dysregulation of IL-2 and IL-8 production in circulating T lymphocytes from young cystic fibrosis patients

It is well documented that patients with cystic fibrosis (CF) are unable to clear persistent airway infections in spite of strong local inflammation, suggesting a dysregulation of immunity in CF. We and others have reported previously that T lymphocytes may play a prominent role in this immune imbalance. In the present work, we compared the reactivity of CD3+ T cells obtained from young CF patients in stable clinical conditions (n = 10, aged 9–16·5 years) to age‐matched healthy subjects (n = 6, aged 9–13·5 years). Intracellular levels of interferon (IFN)‐γ, interleukin (IL)‐2, IL‐8 and IL‐10 were determined by flow cytometry after whole blood culture. The data identified T lymphocyte subsets producing either low levels (M1) or high levels (M2) of cytokine under steady‐state conditions. We found that the production of IFN‐γ and IL‐10 by T lymphocytes was similar between young CF patients and healthy subjects. In contrast, after 4 h of activation with PMA and ionomycin, the percentage of T cells producing high levels of IL‐2 (M2) was greater in CF patients (P = 0·02). Moreover, T cells from CF patients produced lower levels of IL‐8, before and after activation (P = 0·007). We conclude that a systemic immune imbalance is present in young CF patients, even when clinically stable. This disorder is characterized by the capability of circulating T lymphocytes to produce low levels of IL‐8 and by the emergence of more numerous T cells producing high levels of IL‐2. This imbalance may contribute to immune dysregulation in CF.

Involvement of tumor necrosis factor-α during infection of human monocytic cells by Toxoplasma gondii

Abstract Although the involvement of tumor necrosis factor-α (TNF-α) during Toxoplasma gondii infection has largely been described in mouse models, only a few studies in human models have been reported. We demonstrated the role of TNF-α during the process of invasion by T. gondii in human monocytic cells. Cotreatment of cells with interferon-γ (IFN-γ) and lipolysaccharide (LPS) during T. gondii infection induced TNF-α production and decreased the number of parasitized cells (invasion index). Neutralization of the production of TNF-α using a specific monoclonal antibody or pentoxifylline enhanced the invasion index. The relationship between TNF-α production and protection of monocytic cells against T. gondii invasion was confirmed by treatment of infected cultures with exogenous TNF-α. Thus, in contrast to the results obtained in murine models but in accordance with those observed in other human models, the present study shows for the first time in human monocytic cells that T. gondii does not induce any TNF-α secretion and inhibits TNF-α production induced by IFN-γ/LPS.

Regulation of tumor necrosis factor alpha and its specific receptors during Toxoplasma gondii infection in human monocytic cells

Abstract. The coccidian Toxoplasma gondii is an obligate intracellular parasite which can infect all cell types. Among the cytokines elicited by the immune response to Toxoplasma, tumor necrosis factor alpha (TNF-α) acts synergistically with gamma interferon (IFN-γ) and plays a major role in host cell resistance. We have previously reported that TNF-α production induced by IFN-γ/LPS decreases after T. gondii infection of human myelomonocytic THP-1 cells. Here, we investigated the regulation of TNF-α and its specific receptors during T. gondii infection of THP-1 cells. We found that TNF-α production was regulated at a post-transcriptional level and that TNF receptor expression was regulated at a pretranscriptional level. The TNF receptor I shedding and the fall in TNF-α levels observed after T. gondii infection would thus be induced by a parasite component with serine protease activity. These findings indicate that T. gondii participates not only in controlling the cytotoxic effects of TNF-α during the infection process, but also in signal transduction mediated mainly by TNF receptor I.

Multiparameter fluorescence imaging for quantification of TH-1 and TH-2 cytokines at the single-cell level

Immune responses are strongly influenced by the cytokines following antigenic stimulation. Distinct cytokine-producing T cell subsets are well known to play a major role in immune responses and to be differentially regulated during immunological disorders, although the characterization and quantification of the TH-1/TH-2 cytokine pattern in T cells remained not clearly defined. Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. The aim of this study was (1) to quantify the cytokine expression in T cells at the single cell level using optical imaging, (2) and to analyze the influence of cyclic AMP- dependent signal transduction pathway in the balance between the TH-1 and TH-2 cytokine profile. We attempted to study several cytokines (IL-2, IFN-(gamma) , IL-4, IL-10 and IL-13) in peripheral blood mononuclear cells. Cells were prestimulated in vitro using phytohemagglutinin and phorbol ester for 36h, and then further cultured for 8h in the presence of monensin. Cells were permeabilized and then simple-, double- or triple-labeled with the corresponding specific fluorescent monoclonal antibodies. The cell phenotype was also determined by analyzing the expression of each of CD4, CD8, CD45RO and CD45RA with the cytokine expression. Conventional images of cells were recorded with a Peltier- cooled CCD camera (B/W C5985, Hamamatsu photonics) through an inverted microscope equipped with epi-fluorescence (Diaphot 300, Nikon). Images were digitalized using an acquisition video interface (Oculus TCX Coreco) in 762 by 570 pixels coded in 8 bits (256 gray levels), and analyzed thereafter in an IBM PC computer based on an intel pentium processor with an adequate software (Visilog 4, Noesis). The first image processing step is the extraction of cell areas using an edge detection and a binary thresholding method. In order to reduce the background noise of fluorescence, we performed an opening procedure of the original image using a structuring element. The opened image was therefore subtracted from the original one, and the gray intensities were subsequently measured. Fluorescence intensities are mapped in MD representation using Matlab software. Consequently, quantitative comparative expression of intracellular cytokines and cell membrane markers was achieved. Using this technique, we showed that CD4+ and CD8+T lymphocytes expressed a large panel of cytokines, and that protein kinase A (PKA) activation pathway induced a polarization of activated human T cells to the TH-2 type profile. Data also showed different sensitivities of CD45 RO/CD45RA lymphocytes to the activation of PKA, thus suggesting the implication of memory CD4+- and CD8+-T cells in the T cell specific immune and inflammatory processes and their control by PKA activation pathway. Finally, this method represents a powerful tool for the detection and quantification of intracellular cytokine expression and the analysis of the functional properties of T lymphocytes during immune responses.