Ineffective elimination of Leishmania major by inflammatory (MRP14-positive) subtype of monocytic cells.

Myeloid-related protein (MRP) 14, an intracellular protein involved in calcium-dependent activation of myeloid cells, presents a differentiation marker for a subtype of macrophages. In experimental leishmaniasis, BALB/c mice succumb to visceral dissemination after infection with L. major, due to a Th2 cell response, while C57Bl/6 mice develop protective immunity associated with a Th1 cell response. We have previously shown that resistance in (C57Bl/6 mice was also associated with a significantly lower percentage of MRP14-positive cells in the infiltrate than in susceptible BALB/c mice. In C57Bl/6 mice, weekly injections of bone marrow (BM) cells enriched with MRP14-positive cells (d1 of culture) did not reverse, but prolonged the course of infection, associated with increased local parasite spread. In BALB/c mice a single dose of an antiphlogistic agent (dexamethasone or lipoxygenase inhibitor) was associated with reduction of infiltrating MRP14-positive cells and also with a decrease of parasite loads in footpads, lymph nodes as well as spleens, and with delayed progression of disease, Double labeling experiments in vitro revealed that at least 43.1% of MRP14-positive mononuclear cells in BM cultures (8h) had phagocytosed parasites after 4 h of co-incubation. Activation by IFN-gamma (20 U/ml) for 24h and 48h did not significantly reduce parasite load in these cells. In contrast, 77.0% of F4/80-positive macrophages (6d of culture) were infected with L. major parasites and these cells responded to activation with IFN-gamma (20 U/ml) with significant reduction of parasite load (25.3%). The protein MRP14 did not have an effect on parasite survival in vitro. Thus, the impaired capability of MRP14-positive cells to kill L. major upon stimulation may be one reason for the adverse course of infection observed with their increased appearance.

Role of p53 in UVB-induced apoptosis in human HaCaT keratinocytes.

Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged cells. Because the product of the tumor suppressor gene p53 has been demonstrated to be crucial for the induction of apoptosis in certain cell types, the present study was aimed at elucidating its role in ultraviolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultraviolet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time- and concentration-dependent fashion. This increase could not be inhibited by the protein synthesis inhibitor cycloheximide. Because HaCaT keratinocytes are known to bear two p53 point mutations and because it is unclear whether p53 in HaCaT cells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the expression vector pCMV-Neo-Bam in sense (pC53-SN3) and anti-sense (pC53-ASN) direction. After selection with geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polymerase chain reaction. Cell clones bearing the anti-sense product were further analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing cell clones compared with the parental HaCaT cells. These studies demonstrate that blocking mutated p53 can partially block apoptosis in HaCaT keratinocytes and furthermore can confirm the key role for p53 in ultraviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT keratinocytes and their p53-transfectants provide a convenient model that allows for further detailed analyses of apoptosis-associated biochemical and molecular events in human keratinocytes.

E-selectin expression in experimental models of inflammation in mice.

E-selectin (CD62E, formerly termed ELAM-1) is a cytokine-inducible adhesion molecule which mediates the binding of neutrophils, monocytes, and skin homing T-cells. The murine homologue of E-selectin has been cloned. A monoclonal antibody (21KC10) was used here to study immunohistochemically the expression and regulation of murine E-selectin in vitro and in vivo. As described for the human system, there was no staining of normal endothelium in skin and other tissues. LPS and tumour necrosis factor-alpha (TNF-alpha), but not interleukin-4 (IL-4) or interferon-gamma (IFN-gamma), induced a transient expression of E-selectin, both when injected in vivo and when added to endothelial cell lines in vitro. To analyse temporal expression of E-selectin under pathophysiological conditions in vivo, we chose two murine models of inflammation: allergic (ACD) and irritant contact dermatitis (ICD). Expression of E-selectin was found to be induced on vascular endothelium of post-capillary venules in both ACD and ICD. In ICD, maximal staining of endothelial cells occurred earlier than in ACD. Expression of E-selectin during ICD and ACD was then compared between strains of mice which differ with regard to the intensity of their inflammatory reaction. BALB/c mice, which in contrast to C57BI/6 mice show a denser infiltrate and prolonged influx of granulocyte and monocytes, revealed a more pronounced and more prolonged expression of E-selectin than C57BI/6 mice. This held true for both ACD and ICD, and in each case, peak expression of E-selectin was associated with the highest density of the leukocytic infiltrate. This study thus reveals regulatory mechanisms involved in the expression of murine E-selectin in vivo and in vitro. It also demonstrates a correlation between endothelial expression of E-selectin and the genetically determined intensity of the inflammatory response.

Induction of apoptosis in human HaCaT keratinocytes.

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-gamma caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of 'nonapoptotic programmed cell death'. Dexamethasone, tumor necrosis factor-alpha, transforming growth factor-beta, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A2 analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body.

Activated T cells induce expression of E-selectin in vitro and in an antigen-dependent manner in vivo.

E-selectin is an endothelial adhesion molecule for polymorphonuclear cells, monocytes and skin-homing T cells. We have analyzed whether murine T cells are able to induce expression of E-selectin in vitro and in vivo. Using models of inflammation in which T cells play either a significant or only a minor role, we compared induction of E-selectin between normal mice and mice lacking functional T cells (athymic nude mice). In irritant contact dermatitis, a model without a major role for T cells, E-selectin was transiently expressed within the first 24 h in both normal and nude mice. In experimental leishmaniasis (where specific T cells play an important role), a high expression of E-selectin was maintained for 48 h in normal mice, whereas in nude mice expression was only transient. However, reconstitution of nude mice with 10(8) T cells from draining lymph nodes (LN) of Leishmania-infected normal mice could restore sustained expression of E-selectin. Transfer of T lymphocytes from normal LN or from LN of mice sensitized to the contact allergen trinitrochlorobenzene (TNCB) did not have this effect. T cells from TNCB-sensitized mice, however, did induce sustained expression of E-selectin in nude mice when TNCB was applied locally; here, reconstitution with Leishmania-specific T cells had no effect. In vitro, T cells from infected or TNCB-sensitized normal mice increased expression of E-selectin on microvascular endothelial cells after 4 h of co-culture. T cells from untreated mice were less effective. Induction was dependent on direct cell-cell contact, but not on the action of interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha or interferon-gamma. We conclude that sensitized T cells induce sustained expression of E-selectin in vivo in an antigen-dependent manner. This novel way of regulation could be relevant for cell-mediated immunity and chronic disease. The mechanisms are unknown, but, as in vitro, might require direct cell-cell contact.

Expression of murine VCAM-1 in vitro and in different models of inflammation in vivo: correlation with immigration of monocytes.

VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vitro, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism, and the subsesquent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8-positive lymphocytes was only observed in ACD and Leishmaniasis.

Expression of murine VCAM-1 in vitro and in different models of inflammation in vivo: correlation with immigration of monocytes.

VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vivo, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism and the subsequent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8-positive lymphocytes was only observed in ACD and Leishmaniasis.

Substance P and calcitonin gene-related peptide modulate leukocyte infiltration to mouse skin during allergic contact dermatitis.

The effects of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) on leukocyte infiltration during allergic contact dermatitis (ACD) in mice were studied. Concomitant topical application of SP or CGRP with the allergen oxazolone resulted in enhanced leukocyte recruitment at the sites of challenge. Immunohistochemical studies revealed that the numbers of T-helper (L3T4+) and cytotoxic (Lyt-2) lymphocytes and infiltrating macrophages (BM8+) were increased. In addition, ICAM-1 and MHC class II molecule expression by these cells was enhanced after neuropeptide application. Analysis by confocal laser scanning microscopy revealed an increase in the immunoreactivity for SP and CGRP in nerve fibres during the course of ACD. Flow cytometry studies showed that SP and CGRP did not upregulate expression of the adhesion molecules ICAM-1 and VCAM-1 by murine endothelial cell lines in vitro. This suggests that increased infiltration of leukocytes during ACD is not a consequence of direct neuropeptide-promoted upregulation of endothelial adhesion molecules in vivo. In conclusion, our observations provide evidence for a modulatory role of neuropeptides in the pathogenesis of ACD.

Expression and complex assembly of calcium-binding proteins MRP8 and MRP14 during differentiation of murine myelomonocytic cells.

In the present study we analyzed expression and biochemical properties of the two recently cloned calcium-binding proteins MRP8 and MRP14, both members of the S-100 family, in murine myelomonocytic cells. Expression of MRPs was found to be restricted to granulocytes and distinct stages of macrophage differentiation when compared to the expression of other markers (Mac-1, F4/80) in transformed macrophage cell lines (M1, RMB.TG, WEHI.TG, J774A, P388D) and resident and exudate peritoneal and tissue macrophages. Similarly, mRNA and protein levels of MRP8/MRP14 in murine bone marrow cells decreased during culture in L cell-conditioned medium. Applying a cross-linking technique, the formation of noncovalently associated heteromeric MRP8/MRP14 complexes of 25, 35, and 48 kd was demonstrated. Our data suggest that MRP8/MRP14 expression and complexation are characteristic for granulocytes and distinct stages of macrophage differentiation in mice. Analysis of the MRP8/MRP14+ phenotype of macrophages may provide further insights into the mechanisms underlying macrophage differentiation.

Effects of divalent cations on lipid flip-flop in the human erythrocyte membrane.

Treatment of human erythrocytes with ionophore A23187 (10 mumol.l-1) and Ca2+ (0.05-0.5 mmol.l-1) or Sr2+ (0.2-1 mmol.l-1) in results in a concentration-dependent acceleration of the transmembrane reorientation (flip) of the lipid probes lysophosphatidylcholine and palmitoylcarnitine to the inner membrane leaflet after their primary insertion into the outer leaflet. Mg2+, Mn2+, Zn2+ and La3+ do not accelerate flip. Ca2(+)-induced flip acceleration depends also on the ionophore concentration. It is reversed by removal of Ca2+ with EDTA. A causal role of Ca2(+)-induced membrane protein degradation and decrease of the polyphosphoinositide level in flip acceleration could be excluded. Likewise, calmodulin-dependent processes are probably not involved since the calmodulin antagonist calmidazolium (2-10 mumol.l-1) does not suppress but even enhances the Ca2(+)-induced flip acceleration. The same is true for the Ca2+ antagonist flunarizine. These drugs do not alter flip rate in the absence of Ca2+. At high Ca2+ (1-5 mmol.l-1) an initial flip acceleration is followed by flip normalization. High concentrations of Mn2+ and Mg2+ slow down flip rates. The selective acceleration of flip by Ca2+ and Sr2+ is discussed to be due to a local detachment of the membrane skeleton from the bilayer, whereas the unselective slow down of flip by divalent cations might be due to a stabilization of the membrane bilayer by the cations. After loading of cells with Ca2+ (but not with Mn2+) the inner membrane leaflet phospholipid phosphatidylserine becomes rapidly exposed to the outer membrane surface, as detectable by its accessibility to phospholipase A2 (5 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of transbilayer mobility of a membrane lipid probe accompanies formation of membrane leaks during photodynamic treatment of erythrocytes.

In order to further characterize membrane alterations in human erythrocytes subjected to photodynamic treatment the passive transbilayer mobility of a phospholipid analogue was studied in cells illuminated for various lengths of time in the presence of the photosensitizer, aluminum chlorotetrasulfophthalocyanine. These measurements were combined with the characterization of the membrane leaks for polar solutes occurring under the same conditions with respect to their apparent size, number and ion selectivity. The time-dependent photodynamic enhancement of leaks for K+ as well as choline or erythritol was paralleled by a marked increase of the transbilayer reorientation rate of the amphiphilic lipid probe, palmitoyllysophosphatidylcholine from 0.05% min-1 in native cells to 0.32% min-1 after 60 min illumination. The asymmetric orientation of native phospholipids was not affected by this treatment. The leak permeability proved to be due to the formation of pores with apparent radii of about 0.45 nm after 60 min illumination, and of 0.75 nm after 90 min. The number of pores per cell was calculated to be less than 1, the pores are slightly cation-selective (PK/PCl approximately 3:1). Since photodynamic treatment did not induce lipid peroxidation under the prevailing experimental conditions, protein modification must be the primary cause of both, leak permeability and flip enhancement. Since it is also likely that the leak permeability arises from oxidation of intrinsic membrane proteins, the results raise the interesting possibility that oxidative alteration of intrinsic membrane proteins may lead to enhanced transbilayer mobility of lipids.

Relationship between gramicidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes.

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.