Urszula Wojewodzka

Chemokines, cytokines, and growth factors in keratinocytes and dermal endothelial cells in the margin of chronic diabetic foot ulcers

Keratinocytes and dermal endothelial cells, excluding leukocytes that infiltrate wounds, are the main source of soluble factors regulating healing of skin ulcers. We used immunohistochemistry to analyze the expression of various chemotactic and growth factors and their receptors in the margin of diabetic foot ulcers and in normal nondiabetic foot skin. Our study found significantly elevated expression of transforming growth factor‐β1 (TGF‐β1) and type I TGF‐β receptors (TGFβR1), granulocyte macrophage colony‐stimulating factor (GM‐CSF), and epidermal growth factor (EGF) in keratinocytes in the ulcer margin (p<0.05). Significantly increased expression of monocyte chemotactic protein‐1, GM‐CSF, CXCR1, and TGFβRI and decreased expression of interleukin (IL)‐10, IL‐15, and TGF‐β1 were observed in ulcer dermal endothelial cells (p<0.05). There was a lack of up‐regulation of IL‐8, CCR2A, IL‐10 receptor, GM‐CSF receptor, platelet‐derived growth factors and their receptors, vascular endothelial growth factor and its type II receptor, EGF receptor, insulin‐like growth factor‐1, and nitric oxide synthase‐2 in both KCs and endothelial cells in the ulcer. Finally, there was a lack of up‐regulation of IL‐10 and IL‐15 in keratinocytes and of EGF, basic fibroblast growth factor, and nitric oxide synthase‐3 in endothelial cells in the ulcer margins. The enhanced expression of some factors responsible for KC behavior could suggest an unimpaired capacity of keratinocytes to reepithelialize the margin of diabetic foot ulcers. However, lack of up‐regulation of some angiogenic and leukocyte chemotactic factors, associated with the reduced influx of immune cells, may account for a poor formation of granulation tissue and chronicity of ulcer epithelialization.

Reactivity of antibodies directed against human antigens with surface markers on canine leukocytes

A panel of anti-human antibodies was used in order to investigate their cross-reactivity with canine leukocytes. The labeling was carried out at the microscopic level by immunocytochemical staining. Of 50 antibodies 22 cross-reacted with canine leukocytes from afferent lymph and peripheral blood. All leukocytes reacted with MHM23 (CD18). Two anti-HLA DR antibodies, DK22 and L243, reacted mostly with veiled cells and with PHA-stimulated lymphocytes, whereas TAL1B5 was cross-reactive with all canine lymphocytes. The activation markers CD25, Ki-67, PCNA were identified on PHA-stimulated lymphocytes with ACT1, Ki-67 and PC10 clone produced antibodies. Canine eosinophils reacted with MHM6 (CD23) antibody. A large number of antibodies reacted with canine lymph veiled cells. Canine granulocytes and a subset of lymphocytes were stained with the anti-CD15 antibody only after treatment of cytospins with neuraminidase.

Keratinocyte and dermal vascular endothelial cell capacities remain unimpaired in the margin of chronic venous ulcer

The role of endogenously produced cytokines and growth factors in the impaired healing of chronic leg ulcers remains uncertain. The aim of this study was to determine the functional capacity of skin cells in ulcer bed tissue compared to those in the edge of ulcers and skin distal to ulcers. Biopsies from leg ulcers of ten randomly selected patients were examined immunohistochemically for cytokines and growth factors produced by keratinocytes (KC) and vascular endothelial cells (EC). The phenotype of leukocytes infiltrating venous ulcers and the expression of vascular adhesion molecules responsible for extravasation were also studied. The expression of cytokines and growth factors by KC was similar in areas adjacent and remote from an ulcer. In the dermis adjacent to an ulcer, the expression of IL-1α, IL-1β, IL-1Ra, EGF and PDGFa by EC was higher than the levels of expression in EC from the distant dermis. The expression of IL-6, TNFα and GM-CSF was comparable to that in cells from intact dermis. For all these factors staining was cytoplasmic, suggesting production in these areas. Ulcer bed tissue contained few fibroblasts and blood capillaries showing a high staining intensity for CD62E and CD106 EC adhesion molecules but no FGF2 expression (P<0.05). The intensity of staining for scavenging CD15+elastase+ granulocytes and CD35+ (C3bR) activated macrophages in the ulcer bed was comparable to that in the margin but higher than that in the distant dermis (P<0.05), whereas staining for CD68+, HLA DR+, TGFβ+ and CD54+ dermal macrophages was similar in all areas. There was reduced staining for CD4+ and CD8+ cells in the ulcer bed (P<0.05). There were no CD1a+ Langerhans cells in the epidermis encroaching upon the granulation tissue and there was reduced CD1a staining in the adjacent epidermis (P<0.05). In conclusion, there is chronic accumulation of scavenging cells with lack of remodeling of the granulation tissue and, at the same time, preserved cytokine and growth factor secretory potential of KC and dermal EC in non-healing venous leg ulcers.

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells

Smac/DIABLO, a pro‐apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT‐induced apoptosis in living MCF‐7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m‐calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax‐dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.

Translocation of Bax and Bid to mitochondria, endoplasmic reticulum and nuclear envelope: possible control points in apoptosis.

The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.

Expression of apoptosis-related proteins in involuting mammary gland of sow.

The expression of apoptosis-related proteins: TGF-beta1 (local inductor), TGF-beta-receptor, Bax (promoter), Bcl-2 (inhibitor) and CPP-32 (executor of apoptosis); the subcellular distribution of Bax; as well as the number and morphology of apoptotic cells in low-, moderate-, and high-involuted mammary glands of sow (four to six days after weaning) were investigated. The immunohistochemical study demonstrated a statistically significant increase in the integrated optical density (IOD) of lobuloalveolar mammary tissue labelling with anti-Bax antibody from low- through moderate-, to high-involuted glands. The immunoelectron microscopy revealed that Bax was localised in the cytosol, on the membranes of mitochondrium and rough endoplasmic reticulum, in nuclear envelope pores, and over heterochromatin of mammary epithelial cells. The increase in Bax/Bcl-2 ratio (2.3, 2.6 and 5.6 for low-, moderate-, and high-involuted glands, respectively) indicated the increasing susceptibility of mammary epithelial cells to apoptosis in the course of involution. The highest Bax/Bcl-2 ratio in high-involuted glands coincided with the highest expression of CPP-32 (caspase 3), TGF-beta1 and TGF-beta1 receptor. The number of apoptotic cells (simultaneous TUNEL and Hoechst 33342 staining) was 2.7, 3.4 and 3.8% for low-, moderate-, and high-involuted glands, respectively. The ultrastructural evaluation showed characteristic morphological features of apoptosis such as: margination and condensation of chromatin; pyknosis and fragmentation of the nucleus; and formation of apoptotic bodies. Phagocytosis of apoptotic cells by macrophages was also documented. The results of the present study suggest the involvement of Bax/Bcl-2 check-point in the regulation, CPP-32 in the execution, but TGF-beta1 in the induction of apoptosis of mammary epithelial cells in the involuting mammary gland of sow.

CYTOKINES AND ADHERENCE MOLECULES INVOLVED IN SPONTANEOUS DENDRITIC CELL-LYMPHOCYTE CLUSTERING IN SKIN AFFERENT LYMPH

The skin afferent lymph dendritic cell (DC) spontaneously forms clusters with autologous T cells. The role of adhesion molecules and cytokines in this process was investigated. Analysis of the expression of adhesion receptors on the canine peripheral lymph DC revealed the presence of CD54, CD58, CD18 as well as CD49d and CD49e molecules and cell surface fibronectin. The CD54 and CD58 molecules were found to play a key role in the ‘spontaneous’ lymph cell clustering. Antibody against fibronectin, a substrate for CD49d and CD49e receptors, reduced DC‐lymphocyte binding. Analysis of the effect of cytokines revealed that the pro‐inflammatory IL1β rather than ILlα, and TNFα may be responsible for the enhanced lymph cell in vitro clustering. The IL6 had no such augmenting effect. The enhancing effect of endogenous ILlβ present in lymph was reduced by the ILlβ neutralizing antibody. The effect of exogenously added ILlβ was also limited by the IL1 receptor antagonist. The ILlRa alone had no effect on cell binding, even when used in the high doses. Neutralizing of ILlRa in lymph with the specific antibody brought about augmented cluster formation. The enhancing properties of TNFα on cell binding were reduced by the TNFα neutralizing antibody. The IL10 significantly limited lymph DC cluster formation with T cells. In conclusion, these data demonstrate that the present in lymph IL1β and TNFα may be responsible for the observed in vitro enhanced cluster formation of lymph DC with autologous T lymphocytes. Cell binding can be reduced by ILlRa and by IL10. It provides insight into the potential clinical use of these inhibitors.

Growth hormone cell phagocytosis in adenohypophysis of mosaic mice: Morphological and immunocytochemical electron microscopy study

An electron microscopy immunocytochemical study was performed to determine the expression pattern of growth hormone (GH) in mosaic mutant mice adenohypophysis. In normal condition GH was restricted to the secretory granules of all growth hormone cells. Mosaic mice adenohypophysis contained growth hormone cells which have distinctive GH labeled secretory granules at the level seen in control animals. Ultrastructurally, some GH cells of mosaic mice presented abnormalities, but labeling intensity of secretory granules in these cells was always comparable to the basal condition. The striking findings presence of two forms (simple and activated) of folliculo-stellate cells (FS) in close association trough gap or tight junction with GH cells localized especially near the perivascular space. Frequently, in cytoplasm of FS cells, large clusters containing fragments of GH labeled cell were present. Additionally, the existence of large intracellular, electron-lucent spaces, with remnant cellular material in parenchyma of mosaic mutant mice adenohypophysis could suggest intensive process of GH-cell destruction. Our electron microscopy immunocytochemical results provide evidence for loss of GH cells in mosaic mice by phagocytosis. We suppose that impaired body growth observed in mosaic mutant male rats may be, at least partially, a consequence of an alteration in somatotropic axis activity. Loss of GH cells in mosaic mice by phagocytosis supported by FS cells may contribute to this effect.

Liver sinusoidal lymphocytes: their immune functions

Abstract  Recent studies strongly suggest that the liver plays an im portant immunoregulatory role. Ev idence of its role in general immune responsiveness originates from ob servation that, in recipients of liver grafts, the survival of other allo‐grafts is significantly prolonged. The question arises as to which blood lymphocyte subsets, most likely to be responsible for this phenomenon, marginate in liver sinusoids. To study this problem, a liver ex vivo perfusion model was designed for rats. In situ W/WAG livers were wa shed clear of sinusoidal marginating cells prior to and after 1 h perfusion with syngeneic blood. The number of blood cells retained in liver sinu soids, their phenotypes, the respon siveness to mitogen (PHA, 90 μg/ ml) and cytotoxicity against YAC‐1 tumour cells were examined. Our studies showed that rat liver retains in the sinusoids a population of blood cells, enriched in NK, CD8′ and MHC class II+ cells, displaying a high cytotoxic activity and low re sponsiveness to mitogen stimula tion, with a capacity of about 106 cells/g of tissue.

Factors Affecting Spontaneous Dendritic Cell-Lymphocyte Clustering in Skin Afferent Lymph

Dendritic cells (DC) are essential accessory cells for the growth of T lymphocytes and there are evidences that DC start to migrate to regional lymph nodes after contact with antigen. Skin afferent lymph freshly drawn from lymphatics contains DC and lymphocytes (LY), and 3–6% of DC form clusters with LY 1. Direct contact between these cells seems to be an integral part of their interaction in vivo. Clustering is the first phase of antigen presentation to LY and modulation of lymph cell cooperation may prove useful in mitigating skin immune response. In this study we demonstrate factors affecting the “spontaneous” binding of DC with autologous LY in their own environment, that is the lymph.