T-cell-pre-stimulated monocytes promote neovascularisation in a murine hind limb ischaemia model.

AIM: Monocytes play a significant role in neovascularisation. The stimuli that differentiate monocytes along a pro-angio-/arteriogenic-supporting pathway are currently unclear. We investigated whether pre-stimulation of human monocytes with soluble T-cell-derived factors improves revascularisation in murine hind limb ischaemia as a new option for therapeutic angio- and arteriogenesis. DESIGN: Human monocytes were cultured with or without soluble T-cell-derived factors. Unstimulated and pre-stimulated monocytes were transfused after induction of hind limb ischaemia in nude mice. METHODS: Blood flow was measured with laser Doppler perfusion imaging. Collaterals were visualised by immunohistochemistry and angiography. Monocytes were characterised by flowcytometry and Bio-Plex assays. RESULTS: Transfusion of T-cell-pre-stimulated monocytes significantly improved blood flow recovery after hind limb ischaemia and increased collateral size and collateral and capillary number in the post-ischaemic paw. Pre-stimulated monocytes produced a wide variety of factors that support neovascularisation such as platelet-derived growth factor-BB, vascular-endothelial growth factor, interleukin-4 and tumour necrosis factor-α. Few transfused human cells were detected in the muscle tissue, suggesting that paracrine rather than direct effects appear responsible for the enhanced recovery of blood flow observed. CONCLUSION: These results show a beneficial role for T-cell-pre-stimulated monocytes in neovascularisation, rendering the monocyte a potential candidate for regenerative cell therapy that promotes revascularisation in peripheral arterial disease patients.

Macrophages in the murine pancreas and their involvement in fetal endocrine development in vitro.

Macrophages are a heterogeneous population of cells that belong to the mononuclear phagocyte system. They play an important role in tissue homeostasis and remodeling and are also potent immune regulators. Pancreatic macrophages are critically involved in the development and pathogenesis of autoimmune diabetes. To elucidate the ontogeny of pancreatic macrophages, we characterized in this study the macrophages present in the adult and developing fetal pancreas of normal mice. We additionally examined the presence of local macrophage precursors and the involvement of macrophages in the growth of endocrine tissue in the fetal pancreas. We identified two phenotypically distinct macrophage subsets in the adult pancreas. The majority of macrophages was CD45(+)ER-MP23(+)MOMA-1(+). Under noninflammatory conditions, only a minority ( approximately 5%) of the pancreatic macrophages additionally expressed the macrophage marker F4/80. In contrast, in the fetal pancreas, phenotypically, mature macrophages were identified exclusively by their expression of F4/80 and lacked detectable staining with ER-MP23 and MOMA-1 antibodies. In fetal pancreas organ cultures, we could show that macrophages develop from pre-existing precursors, which are present in the fetal pancreas at embryonic age 12.5. Moreover, the number of macrophages increased significantly when macrophage-colony stimulating factor was added to these cultures. It is important that this increase of F4/80-positive cells was paralleled by an increase in the number of insulin-producing cells, suggesting that macrophages support the growth of these endocrine cells.

Fibronectin receptor defects in NOD mouse leucocytes: possible consequences for type 1 diabetes.

Integrins of the very late antigen (VLA) family mediate leucocyte traffic to lymphoid organs under physiological conditions and in chronic inflammatory situations such as autoimmunity. Accordingly, the current thinking is of a positive correlation between VLA expression and capability of the generation of autoimmunity. Herein we discuss recent findings on the defective expression of integrin-type fibronectin receptors alpha4beta1 (VLA-4) and alpha5beta1 (VLA-5) in the non-obese diabetic (NOD) mouse, a murine model of autoimmune insulin-dependent diabetes mellitus. As compared with normal animals, NOD thymocytes (including the CD4+CD25+ regulatory T cells) exhibit a decrease in the membrane expression of alpha5beta1, resulting in a functional impairment of fibronectin-mediated interactions, including cell migration. Interestingly, thymocytes that are trapped within the giant perivascular spaces seen in NOD thymus are consistently alpha5beta1 negative, suggesting that the progressive arrest of mature cells can be related to the alpha5beta1 defect. Peripheral T cells also exhibit decreased alpha5beta1 membrane expression and impaired fibronectin-driven migration. Additionally, we observed a defect in alpha4beta1 fibronectin receptor expression in NOD macrophages. Peritoneal, bone marrow-derived-precursor, as well as thymic macrophages of NOD mice showed an impaired upregulation of alpha4-integrin chain expression, dependent on the level of macrophage maturation. Overall these data lead to the notion that NOD leucocytes bear distinct fibronectin receptor-mediated cell migration defects, which may be involved in the pathogenesis and/or pathophysiology of the autoimmune events seen in NOD mice. Further studies will be helpful to define whether or not this concept can be applied for other autoimmune diseases.

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells.

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 plaque-forming units of the hlL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors responded. However, intratumoral treatment with this vector significantly enhanced tumor regression induced by systemic IL-2 protein treatment, which was used as a positive control. In addition we show, by performing delayed-type of hypersensitivity assays, that AdCnull when injected intratumorally enhances recognition of tumor antigens by T lymphocytes to the same extent as intratumoral treatment with the IL-2-expressing vector. The replication-deficient adenoviruses appear to have a therapeutic advantage in cytokine-mediated immunotherapy: even adenovirus vectors that do not express a transgene, show adjuvant activity and stimulate an antitumor immune response.

Differential expression of stress proteins in human adult astrocytes in response to cytokines.

Various lines of evidence suggest a close relationship between heat shock proteins (hsp) and several autoimmune diseases such as arthritis, diabetes and multiple sclerosis. While enhanced expression of hsp in autoimmune diseases is often regarded as a non-specific bystander effect of the inflammatory process, surprisingly little is known on hsp regulation by inflammatory mediators such as cytokines. In this study cytokine-induced expression of hsp60, hsp27 and alphaB-crystallin was studied in cultures of primary human adult astrocytes at the mRNA as well as at the protein level. We show differential hsp expression patterns in response to pro-inflammatory and immunoregulatory cytokines. Hsp60 expression was found to be enhanced in response to cytokines as diverse as IL-1beta, TNF-alpha, IL-4, IL-6 and IL-10. Upregulation of hsp27, however, was primarily induced by immunoregulatory cytokines like IL-4, IL-6 and TGF-beta whereas alphaB-crystallin expression was found to be enhanced by the pro-inflammatory cytokine TNF-alpha only. None of the cytokines studied was able to enhance expression of all three hsp simultaneously. These results show that in human astrocytes induced expression of hsp27 and alphaB-crystallin is dependent on the presence of a defined set of stimuli, while induced expression of hsp60 is a much less selective event. This highly differential pattern of hsp expression in response to inflammatory mediators known to play an important role in the pathogenesis of autoimmune diseases indicates that hsp responses are specific rather than non-specific bystander responses.

The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alphaB-crystallin, Hsp27, and Hsp60.

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.

EBV-induced expression and HLA-DR-restricted presentation by human B cells of alpha B-crystallin, a candidate autoantigen in multiple sclerosis.

The development of multiple sclerosis is most likely influenced by autoimmune responses to central nervous system myelin proteins as well as by infections with common viruses such as EBV and human herpesvirus-6. However, much remains to be established on how these factors interact. In this study, we show that upon EBV infection, human B cells start to express alpha B-crystallin, a small stress protein that was identified previously as an immunodominant Ag of CNS myelin in multiple sclerosis patients. EBV-induced expression of alpha B-crystallin in B cells leads to HLA-DR-restricted presentation of the protein and to activation of proinflammatory alpha B-crystallin-specific Th cells. While alpha B-crystallin is present in EBV-infected human B cells, the protein is absent from human lymphoid tissues under normal conditions. This is in sharp contrast to other stress proteins such as heat-shock protein (hsp)27 and hsp60 that are ubiquitously expressed in these tissues. In addition, the absence of alpha B-crystallin from lymphoid tissues in humans is unique as compared with other mammals. All other species examined, including rodents, sheep, and primates, showed constitutive expression of alpha B-crystallin in secondary lymphoid tissues and sometimes even in the thymus. Since constitutive lymphoid expression most likely results in immunologic tolerance, such a state of tolerance to alpha B-crystallin can be expected for all of these species, but not for humans. When taken together, our data provide evidence for a novel mechanism by which common viral infections can trigger myelin-directed autoimmunity in a way that is unique for humans.