Macrophage migration inhibitory factor counterregulates dexamethasone-mediated suppression of hypoxia-inducible factor-1 alpha function and differentially influences human CD4+ T cell proliferation under hypoxia.

Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary human CD4(+) T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4(+) T cells. In this article, we show that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4(+) T cells, as well as in Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1α expression and HIF-1 target gene induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation. Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-mediated suppression of MIF and HIF-1α expression. Based on these data, we suggest that hypoxia significantly affects the expression of HIF-1α in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4(+) T cells, thus influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF could be useful targets to optimize GC therapy when treating inflammation.

Human early fracture hematoma is characterized by inflammation and hypoxia.

BACKGROUND: An effective immune system, especially during the inflammatory phase, putatively influences the quality and likelihood of bone healing. If and how this is reflected within the initial fracture hematoma is unclear. QUESTIONS/PURPOSES: We therefore asked the following questions: (1) Does the local expression in fracture hematoma of genes involved in adaptation to hypoxia, migration, angiogenesis, and osteogenesis vary as compared to the peripheral blood? (2) Do these changes occur time dependently? (3) Is the gene expression during fracture hematoma formation altered by irradiation? METHODS: Cells from fracture hematoma of 20 patients and hematomas formed in 40 patients after THA (20 without and 20 with preoperative radiation) were isolated and RNA was extracted to analyze the influence of oxygen deprivation during fracture healing on mRNA expression of genes (HIF1A, LDHA, and PGK1) involved in immunoregulation (IL6, IL8, CXCR4), angiogenesis (VEGF, IL8), and osteogenesis (SPP1, RUNX2) by quantitative PCR. RESULTS: We observed locally increased LDHA gene expression in fracture hematoma cells (6-72 h post fracture) reflecting the adaptation to hypoxia. IL6, IL8, and VEGF upregulation indicated hypoxia-mediated inflammation and angiogenesis; increased CXCR4 expression reflected immigration of immune cells. Osteogenic differentiation was reflected in the increased expression of the SPP1 and RUNX2 genes. The increased expression of the LDHA, VEGF, IL8, SPP1 and RUNX2 genes occurred time dependently. Irradiation suppressed HIF1A, IL6, IL8, CXCR4, and RUNX2 gene expression. CONCLUSIONS: Our data suggest cells in the fracture hematoma (1) adapt to hypoxia and (2) promote inflammation in fracture healing at the mRNA level, indicating early involvement of the immune system. CLINICAL RELEVANCE: The initial fracture hematoma is important for the onset of angiogenesis, chemotaxis, and osteogenesis.

Toxicity of antiseptics against chondrocytes: what is best for the cartilage in septic joint surgery?

In septic joint surgery, the most frequently used antiseptics are polyhexanide, hydrogen peroxide and taurolidine. The aim of this study was to examine the effects of these antiseptics on viability of human chondrocytes. Our hypothesis was that antiseptics and supplemental irrigation with sodium chloride lavage are less toxic on human chondrocytes than treatment with antiseptics only. Primary human chondrocytes were isolated and cultured from six donated human knee joints. Polyhexanide, hydrogen peroxide or taurolidine were added to the cultures. Toxicity analysis was performed by visualisation of cell structure using light microscopy and LDH activity. The determination of vital cells and total cell numbers of chondrocytes treated with antiseptics partly followed by irrigation with sodium chloride solution was performed by using Casy Cell-Counter. Light microscopic data revealed a defect in cell structure after addition of antiseptics. We showed a significant increase of LDH enzyme activity after the treatment with polyhexanide or taurolidine. After treatment with antiseptics followed by sodium chloride solution a significant increase of vital and total cell numbers resulted in comparison with the chondrocytes that were only treated with antiseptics. The data show that treatment with polyhexanid, hydrogen peroxide or taurolidine induces cell death of human chondroctes in vitro. The application of sodium chloride solution after the treatment with polyhexanide and hydrogen peroxide possibly has a protective effect on chondrocyte viability.

CD152 (CTLA-4) regulates effector functions of CD8+ T lymphocytes by repressing Eomesodermin.

CD8(+) T lymphocytes are required for effective host defense against pathogens and also for mediating effector responses against uncontrolled proliferating self-tissues. In this study, we determine that individual CD8(+) T cells are tightly controlled in their effector functions by CD152 (CTLA-4). We demonstrate that signals induced by CD152 reduce the frequency of IFN-gamma and granzyme B expressing CD8(+) T cells independently of the transcription factors T-bet or cKrox by selectively inhibiting accumulation of Eomesodermin mRNA and protein. Ectopic expression of Eomesodermin reversed the CD152-mediated inhibition of effector molecule production. Additionally, enhanced cytotoxicity of individual CD8(+) T cells differentiated in the absence of CD152 signaling was determined in vivo. These novel insights extend our understanding of how immune responses of CD8(+) T cells are selectively modulated.

CTLA-4 (CD152) inhibits T cell function by activating the ubiquitin ligase Itch.

CTLA-4 (CD152) is a regulatory molecule in the immune system fundamentally important for the inhibition of T cell activity that is mediated by an unknown mechanism. Here we demonstrate similarities of CTLA-4 and Itch deficient mice and that CTLA-4 deficient T cells show a massive reduction in the overall ubiquitination of proteins. CTLA-4-mediated signal transduction leads to increased de-phosphorylation and therefore activation of the ubiquitin ligase Itch and enhanced ubiquitination of the Itch target molecule JunB. The knock-down of Itch completely abolishes the inhibitory effect of CTLA-4-mediated signal transduction on mRNA accumulation of IFN-gamma and IL-4. These results show that CTLA-4 mediates signals via the activation of the ubiquitin ligase Itch probably leading to the enhanced ubiquitination of Itch target molecules resulting in inhibition of T cell activity.

The early fracture hematoma and its potential role in fracture healing.

Research regarding the potency and potential of the fracture hematoma has begun to receive increasing attention. However, currently there is a paucity of relevant literature on the capability and composition of the fracture hematoma. This review briefly summarizes the regenerative fracture healing process and the close interplay between the skeletal and immune systems. The role of immune cells in wound healing is also discussed to clarify their involvement in immunological processes during regeneration. We attempt to describe the current state of knowledge regarding the fracture hematoma as the initial stage of the regenerative process of fracture healing. The review discusses how a better understanding of immune reactions in the hematoma may have implications for bone tissue engineering strategies. We conclude the review by emphasizing how additional investigations of the initial phase of healing will allow us to better differentiate between deleterious and beneficial aspects of inflammation, thereby facilitating improved fracture treatment strategies.

CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in mice.

OBJECTIVE: CD4+CD25+ regulatory T cells (known as Treg cells) suppress unwanted and autoreactive T cell responses. Treg cells express the costimulatory molecule CTLA-4 intracellularly, but the mechanisms by which Treg cells exploit CTLA-4 signaling remain unclear. The present study was undertaken to investigate the role of CTLA-4 in controlling the homeostasis and suppressive function of Treg cells. METHODS: Murine Treg cells were analyzed by flow cytometry for coexpression of CTLA-4 and typical Treg cell-expressed molecules, and the influence of CTLA-4 on T cell proliferation, suppression, and apoptosis was investigated by in vitro assays. To analyze the importance of CTLA-4 in Treg cell-mediated suppression in vivo, wild-type Treg cells were transferred into CTLA-4-deficient mice displaying lymphoproliferation, and survival was monitored over time. RESULTS: A strong correlation between expression of forkhead box P3 and ex vivo expression of CTLA-4 in Treg cells was observed. Inhibition of CTLA-4 signaling in Treg cells during in vitro stimulation increased cell cycling and led to enhanced activation-induced cell death (AICD), which was mediated by CD95/CD95 ligand-induced activation of caspases. Blockade of CTLA-4 signaling resulted in impairment of the suppressive capacity of Treg cells. Despite these effects, high amounts of Treg cells persisted in CTLA-4-deficient mice. Results of transfer experiments in CTLA-4-deficient mice showed that the mice had a significantly prolonged lifespan when CTLA-4-competent Treg cells were injected. CONCLUSION: Expression of CTLA-4 on Treg cells serves to control T cell proliferation, to confer resistance against AICD, and to maintain the suppressive function of Treg cells.

Cellular composition of the initial fracture hematoma compared to a muscle hematoma: a study in sheep.

Bone fracture leads to a cycle of inflammation, cellular migration, and proliferation to restore tissue integrity. Immune cells at the site of injury are involved especially in the early phase of the healing process, but little is known about the cells present in the initial fracture hematoma. The hypothesis of this study was that the cellular composition in a fracture hematoma differs from that found in a muscle hematoma and that these divergences get more pronounced over time. By using a reproducible osteotomy model and muscle trauma in sheep the distributions of the immune cell subpopulations were evaluated 1 and 4 h after surgery. The cell amount within the first 4 h increased in both hematoma. The number of dead cells was higher in the muscle hematoma. One hour postoperatively the initial fracture hematoma revealed a lower granulocyte percentage compared to the muscle hematoma. The ratio of T helper to cytotoxic T cells was higher in the fracture hematoma compared to the muscle hematoma at both investigated time points. B cell percentage increased in the fracture but not in the muscle hematoma from 1 to 4 h. This is the first study that compares the immune cell subpopulations of a fracture and muscle hematoma.

CD152 (CTLA-4) determines CD4 T cell migration in vitro and in vivo.

BACKGROUND: Migration of antigen-experienced T cells to secondary lymphoid organs and the site of antigenic-challenge is a mandatory prerequisite for the precise functioning of adaptive immune responses. The surface molecule CD152 (CTLA-4) is mostly considered as a negative regulator of T cell activation during immune responses. It is currently unknown whether CD152 can also influence chemokine-driven T cell migration. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the consequences of CD152 signaling on Th cell migration using chemotaxis assays in vitro and radioactive cell tracking in vivo. We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner. In addition, retroviral transduction of CD152 cDNA into CD152 negative cells restored Th1 cell migration. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Using sensitive liposome technology, we show that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and the CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo. CONCLUSIONS/SIGNIFICANCE: We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge.

Adaptation of human CD4+ T cells to pathophysiological hypoxia: a transcriptome analysis.

OBJECTIVE: Inflamed tissues are usually characterized by low oxygen levels. We investigated whether pathophysiological hypoxia (pO(2) < 1%) as found in the rheumatoid synovium modulates the transcriptome of human CD4+ T cells. METHODS: We analyzed the extent to which hypoxia influences the transcriptome in the rheumatoid synovium according to a gene cluster reflecting adaptation to low oxygen levels. Hypoxia-inducible factor-1alpha (HIF-1alpha) was detected in the rheumatoid synovium using immunohistochemistry. Isolated human CD4+ T cells were exposed to hypoxia and analyzed using microarray analysis, quantitative polymerase chain reaction, and immunoblot detection. RESULTS: In rheumatoid arthritis (RA) synovial tissue samples, hypoxia modulates the transcription profile. This profile is similar, but not identical, to that found in isolated CD4+ T cells incubated under hypoxic conditions. We show that HIF-1alpha is expressed in synovial tissue samples and in hypoxic CD4+ cells; and that hypoxia directly affects differential gene expression in human T cells with up to 4.8% modulation of the transcriptome. Functional genome analysis revealed substantial effects of hypoxia on immune response, transcriptional regulation, protein modification, cell growth and proliferation, and cell metabolism. CONCLUSION: Severe hypoxia, a feature of joint inflammation, considerably modulates the transcriptome of cells found in the rheumatoid synovium. Human CD4+ T cells adapt to hypoxic conditions mainly by HIF-1-driven effects on the transcriptome reflecting a profound influence on immune functions. Thus, hypoxia must be taken into account when therapeutically targeting inflammation.