Trichohyalin-like proteins have evolutionarily conserved roles in the morphogenesis of skin appendages.

S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the epidermis and various skin appendages. To determine evolutionarily conserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their amino acid sequences and expression patterns with those of mammalian SFTPs. We identified an ortholog of cornulin and a previously unknown SFTP, termed scaffoldin, in reptiles and birds, whereas filaggrin was confined to mammals. In contrast to mammalian SFTPs, both cornulin and scaffoldin of the chicken are expressed in the embryonic periderm. However, scaffoldin resembles mammalian trichohyalin with regard to its expression in the filiform papillae of the tongue and in the epithelium underneath the forming tips of the claws. Furthermore, scaffoldin is expressed in the epithelial sheath around growing feathers, reminiscent of trichohyalin expression in the inner root sheath of hair. The results of this study show that SFTP-positive epithelia function as scaffolds for the growth of diverse skin appendages such as claws, nails, hair, and feathers, indicating a common evolutionary origin.

Loss of keratin K2 expression causes aberrant aggregation of K10, hyperkeratosis, and inflammation.

Keratin K2 is one of the most abundant structural proteins of the epidermis; however, its biological significance has remained elusive. Here we show that suprabasal type II keratins, K1 and K2, are expressed in a mutually exclusive manner at different body sites of the mouse, with K2 being confined to the ear, sole, and tail skin. Deletion of K2 caused acanthosis and hyperkeratosis of the ear and the tail epidermis, corneocyte fragility, increased transepidermal water loss, and local inflammation in the ear skin. The loss of K2 was partially compensated by upregulation of K1 expression. However, a significant portion of K2-deficient suprabasal keratinocytes lacked a regular cytoskeleton and developed massive aggregates of the type I keratin, K10. Aggregate formation, but not hyperkeratosis, was suppressed by the deletion of both K2 and K10, whereas deletion of K10 alone caused clumping of K2 in ear skin. Taken together, this study demonstrates that K2 is a necessary and sufficient binding partner of K10 at distinct body sites of the mouse and that unbalanced expression of these keratins results in aggregate formation.

Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

Epidermal keratinocytes form a functional skin barrier in the absence of Atg7 dependent autophagy.

BACKGROUND: Cornification of keratinocytes involves the degradation of intracellular constituents which has led to the hypothesis that autophagy plays a role in this process. Mice, in which essential autophagy-related genes such as Atg7 are deleted systemically, die after birth and have not been characterized for potential epidermal defects. OBJECTIVE: This study tested whether autophagy is essential for epidermal barrier formation and function. METHODS: Atg7 was inactivated in epidermal keratinocytes by the Cre-loxP system under the control of the keratin K14 promoter (Atg7Δepi mice). Autophagic activity was detected using the GFP-microtubule-associated protein light chain 3 (GFP-LC3) reporter construct and Western blot analysis of LC3. Epidermal morphology was examined by histological and ultrastructural analyses, and barrier functions were assessed by dye diffusion and water loss assays. RESULTS: Suprabasal epidermal cells of normal mice contained GFP-LC3-labeled autophagosomes and epidermal lysates of these mice showed an excess of lipidated over non-lipidated LC3. These features of active autophagy were efficiently suppressed in Atg7Δepi epidermis. Atg7Δepi mice survived the perinatal period and were apparently healthy. Histologically, their epidermis was inconspicuous and ultrastructural analysis revealed no significant defect in cornification. There was however, an increase in the thickness of corneocytes in the back skin of mutant mice. Nevertheless, resistance to dye penetration into the skin and transepidermal water loss were normal in Atg7Δepi mice. CONCLUSION: This study demonstrates that autophagy is constitutively active in the epidermis but not essential for the barrier function of the skin.

Essential role of the keratinocyte-specific endonuclease DNase1L2 in the removal of nuclear DNA from hair and nails.

Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.

Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial role of endogenous urocanic acid in photoprotection.

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Primary sources and immunological prerequisites for sST2 secretion in humans.

AIMS: Serum levels of the soluble growth stimulation gene-2 (sST2) are elevated in heart and pulmonary diseases. However, the relationship of the sST2/interleukin (IL)-33 axis and its triggers as well as its organ distribution is still not known. This study was thus designed to investigate the cellular origin and regulation of sST2 and IL-33 in vitro and in vivo. METHODS AND RESULTS: sST2 and IL-33 gene expression and protein secretion were analysed in pooled organ-specific cDNAs and in primary cell cultures, respectively, by RT-PCR and ELISA technology. The strongest sST2 mRNA expression was detected in heart and lung tissues, which correlated with spontaneous secretion of sST2 protein in vitro. The inflammatory cytokines IL-1alpha, IL-1beta, and tumour necrosis factor alpha as well as supernatants of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells led to an enhanced secretion of sST2 in cultured cardiac myocytes and lung alveolar epithelial cells. These cytokines enhanced sST2 secretion via an NFkappaB-dependent mechanism. In addition, LPS stimulation in humans in vivo induced a short-term inflammatory response that was followed by a massive enhancement of sST2 secretion. CONCLUSION: These results identify the primary sources and inflammatory triggers for the enhancement of sST2 secretion and demonstrate a relationship between inflammation and the secretion of a bioactive member of the IL-1R family, both in vitro and in vivo.

The anatomy and development of the claws of Xenopus laevis (Lissamphibia: Anura) reveal alternate pathways of structural evolution in the integument of tetrapods.

Digital end organs composed of hard, modified epidermis, generally referred to as claws, are present in mammals and reptiles as well as in several non-amniote taxa such as clawed salamanders and frogs, including Xenopus laevis. So far, only the claws and nails of mammals have been characterized extensively and the question of whether claws were present in the common ancestor of all extant tetrapods is as yet unresolved. To provide a basis for comparisons between amniote and non-amniote claws, we investigated the development, growth and ultrastructure of the epidermal component of the claws of X. laevis. Histological examination of developing claws of X. laevis shows that claw formation is initiated at the tip of the toe by the appearance of superficial cornified cells that are dark brown. Subsequent accumulation of new, proximally extended claw sheath corneocyte layers increases the length of the claw. Histological studies of adult claws show that proliferation of cornifying claw sheath cells occurs along the entire length of the claw-forming epidermis. Living epidermal cells that are converting into the cornified claw sheath corneocytes undergo a form of programmed cell death that is accompanied by degradation of nuclear DNA. Subsequently, the cytoplasm and the nuclear remnants acquire a brown colour by an as-yet unknown mechanism that is likely homologous to the colouration mechanism that occurs in other hard, cornified structures of amphibians such as nuptial pads and tadpole beaks. Transmission electron microscopy revealed that the cornified claw sheath consists of parallel layers of corneocytes with interdigitations being confined to intra-layer contacts and a cementing substance filling the intercorneocyte spaces. Together with recent reports that showed the main molecular components of amniote claws are absent in Xenopus, our data support the hypothesis that claws of amphibians likely represent clade-specific innovations, non-homologous to amniote claws.

Degradation by stratum corneum proteases prevents endogenous RNase inhibitor from blocking antimicrobial activities of RNase 5 and RNase 7.

The antimicrobial defense of the skin is partially mediated by RNase 7, an abundant ribonuclease of the stratum corneum (SC). Here, we investigated the expression and regulation of members of the RNase A family and of the endogenous RNase inhibitor (RI) protein in epidermal keratinocytes (KCs). Reverse transcription-PCR screening revealed that KCs expressed not only RNase 7 but also RNase 5, which was shown earlier to kill the yeast Candida albicans, as well as RNase 1, RNase 4, and RI. The mRNA and protein levels of RNase 5, RNase 7, and RI increased during KC differentiation. When RNase 5 and RNase 7 were incubated with RI in vitro, not only their ribonucleolytic activities but also their antimicrobial activities were strongly suppressed. Immunochemical analyses revealed that SC contains RNase 5, whereas RI was not detectable. Unlike recombinant RNase 5, recombinant RI was degraded when exposed to SC extract. The addition of aprotinin prevented the degradation of RI, indicating that serine proteases of the SC cleave RI. Taken together, this study adds RNase 5 to the list of antimicrobial factors present in the SC and suggests that proteases contribute indirectly to the defense function of the SC by releasing the RI-mediated inhibition of RNase 5 and RNase 7.

Inactivation of VEGF in mammary gland epithelium severely compromises mammary gland development and function.

To investigate the role of the angiogenic cytokine vascular endothelial growth factor (VEGF) during pregnancy and lactation, we used mice in which VEGF had been inactivated in mammary gland epithelial cells. Pups born to mutant mothers failed to thrive, displaying little milk in their stomachs. However, when they were transferred to control mothers they developed normally. Investigation of the mammary gland morphology revealed that lobulo-alveolar expansion into the fat pad was not complete in lactating mutant glands, and an accumulation of fat globules was evident in their secretory epithelium. In contrast to control glands, lactating mutant glands failed to up-regulate mRNAs for genes involved in milk secretion. Blood vessel density was comparable in pregnant mice of both groups but was only half that of controls in lactating mutant mice. FITC-labeled albumin injected intravenously (i.v.) into lactating mice extravasated rapidly and accumulated in the mammary gland epithelial cells in control animals, but was almost completely retained within the vessels in the mutants. Injection of recombinant VEGF i.v. reversed this effect. These findings demonstrate that mammary epithelium-derived VEGF is partially dispensable for angiogenesis during pregnancy and lactation, and by regulating vascular function during lactation, this factor is crucial to mammary gland differentiation and milk production.

DNase1L2 degrades nuclear DNA during corneocyte formation.

The removal of keratinocyte (KC) nuclear DNA by deoxyribonucleases (DNases) is an important step in the formation of normal stratum corneum (SC). However, the molecular identity of the DNA-degrading enzymes has so far remained elusive. Here we show that the endonuclease DNase1-like 2 (DNase1L2) is preferentially expressed in the epidermis and that its expression correlates with terminal differentiation of KC in vitro and in vivo. In biopsies of normal skin, DNase1L2 mRNA was regularly found in suprabasal KC and DNase1L2 protein was highly abundant in the stratum granulosum. In contrast to normal skin, DNase1L2 expression was downregulated in parakeratotic epidermis such as in psoriatic lesions. When DNase1L2 gene expression was knocked down by small interfering RNA in a human skin equivalent model, nuclei were maintained through all layers of the SC. Taken together, our data demonstrate that DNase1L2 plays an essential role in DNA degradation during terminal differentiation of epidermal KC.

Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis.

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.

Cell surface membrane antigen phenotype of human gastrointestinal mast cells.

BACKGROUND: Mast cells (MC) are important effector cells of allergic and inflammatory reactions in diverse organs. These cells interact with a number of other immune cells and structural cells in the tissues as well as with proinflammatory mediators and cytokines. The various interactions are considered to be mediated through distinct cell surface membrane receptors on MC. METHODS: In the present study, we have established the cell surface membrane phenotype of human gastrointestinal MC (HGMC) using a panel of monoclonal antibodies and indirect immunofluorescence staining techniques. RESULTS: HGMC were found to react with antibodies against CD29, CD33, CD44, CD45, CD47, CD54, CD55, CD58, CD63, CD117, CD147, CD151, CD172a, and CD203c. By contrast, HGMC did not express detectable amounts of CD1, CD2, CD4, CD5, CD14, CD15, CD16, CD22, CD24, CD25, CD26, CD27, CD28, CD31, CD32, CD34, CD35, CD88, or CD116. The alpha-chain of the IL-3 receptor (CD123) was detectable neither in resting HGMC nor in HGMC exposed to stem cell factor and interleukin-4. CONCLUSIONS: HGMC express a unique profile of surface antigens including the receptor for mast cell growth factor, adhesion-related molecules, and activation-linked membrane antigens.

Evaluation of normal and neoplastic human mast cells for expression of CD172a (SIRPalpha), CD47, and SHP-1.

Signal regulatory proteins (SIRPs) and tyrosine phosphatases have recently been implicated in the control of receptor tyrosine kinase (RTK)-dependent cell growth. In systemic mastocytosis (SM), neoplastic cells are driven by the RTK KIT, which is mutated at codon 816 in most patients. We examined expression of SIRPalpha, SIRPalpha ligand CD47, and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), a tyrosine phosphatase-type, negative regulator of KIT-dependent signaling, in normal human lung mast cells (HLMC) and neoplastic MC obtained from nine patients with SM. As assessed by multicolor flow cytometry, normal LMC expressed SIRPalpha, CD47, and SHP-1. In patients with SM, MC also reacted with antibodies against SIRPalpha and CD47. By contrast, the levels of SHP-1 were low or undetectable in MC in most cases. Corresponding data were obtained from mRNA analysis. In fact, whereas SIRPalpha mRNA and CD47 mRNA were detected in all samples, the levels of SHP-1 mRNA varied among donors. To demonstrate adhesive functions for SIRPalpha and CD47 on neoplastic MC, an adhesion assay was applied using the MC leukemia cell line HMC-1, which was found to bind to immobilized extracellular domains of SIRPalpha1 (SIRPalpha1ex) and CD47 (CD47ex), and binding of these cells to CD47ex was inhibited by the CD172 antibody SE5A5. In summary, our data show that MC express functional SIRPalpha and CD47 in SM, whereas expression of SHP-1 varies among donors and is low compared with LMC. It is hypothesized that CD172 and CD47 contribute to MC clustering and that the "lack" of SHP-1 in MC may facilitate KIT-dependent signaling in a subgroup of patients.

Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.

Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

Stratum corneum-derived caspase-14 is catalytically active.

Caspase-14, a cysteine protease with restricted tissue distribution, is highly expressed in differentiated epidermal keratinocytes. Here, we extracted soluble proteins from stratum corneum (SC) of human epidermis and demonstrate that the extract cleaves tetrapeptide caspase substrates. The activity decreased to below 10% when caspase-14 was removed by immunodepletion showing that caspase-14 is the predominant caspase in SC. In contrast to normal SC, where caspase-14 was present exclusively in its processed form, incompletely matured SC of parakeratotic skin from psoriasis and seborrheic dermatitis contained both procaspase-14 and caspase-14 subunits. Fractionation of extract from parakeratotic SC revealed that the peak caspase activity coeluted with processed caspase-14 but not with procaspase-14. Our results suggest that during regular terminal keratinocyte differentiation, endogenous procaspase-14 is converted to caspase-14 subunits that are catalytically active in the outermost layers of normal human skin.

Effect of statins on lipoprotein receptor expression in cell lines from human mast cells and basophils.

OBJECTIVE: Statins are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and widely used to treat hyperlipidaemia. Apart from their direct lipid-lowering effects, statins may also influence lipid metabolism through modulation of low-density lipoprotein (LDL) receptors. Basophils and mast cells have been reported to express LDL receptors and have been implicated in atherogenesis. The aim of this study was to investigate the effects of statins on the interactions of 125I-LDL with purified primary human blood basophils, a human basophil cell line, KU812, and a human mast cell line, HMC-1. METHODS: Direct binding experiments were carried out with the primary basophils and KU812 as well as HMC-1 cells before and after pretreatment of the cells with atorvastatin, simvastatin, or cerivastatin. The effects of these three statins on the LDL-uptake and degradation as well as on thymidine incorporation in the cells were also studied. RESULTS: Primary basophils, HMC-1 and KU812 cells expressed two classes of LDL binding sites. Exposure to atorvastatin, simvastatin or cerivastatin increased significantly ( P<0.05) the number of 125I-LDL binding sites on primary basophils and HMC-1 as well as KU812 cells. The effects of the statins were dose dependent. The statins also enhanced the uptake and degradation of LDL in primary basophils, HMC-1 and KU812 cells. The increase in the number of LDL binding sites induced by statins was abolished by mevalonic acid (200 micromol/l). Statins had no effect on the thymidine incorporation into the cells in an unstimulated condition. CONCLUSION: Our results provide evidence for the upregulation of LDL binding sites on human basophils and mast cells by statins. We hypothesise that effects of statins on the lipid metabolism might also involve basophils and mast cells.

Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia. Correlation between VEGF expression and the FAB category.

We studied vascular endothelial growth factor (VEGF) expression in bone marrow sections obtained from 3 healthy donors and 41 patients with acute myeloid leukemia (AML) of various French-American-British (FAB) subtypes by immunohistochemical analysis using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody reacted with myeloid progenitor cells and megakaryocytes but not with erythroid cells or mature granulocytic cells. High levels of VEGF were found in the bone marrow in patients with AML-M1, -M2, -M3, -M4, -M4Eo, and -M5. In these leukemias, the vast majority of myeloblasts (> 90%) expressed VEGF. By contrast, in AML-M0, the percentage of VEGF-positive blasts was lower in most cases (median, 42%), and if at all detectable, these blast cells contained only trace amounts of VEGF. In AML-M3 and -M4Eo, maturing granulocytes failed to express VEGF similar to granulocytes in normal bone marrow. In AML-M6, myeloblasts exhibited VEGF, whereas erythroid cells did not. In AML-M7, blast cells and megakaryocytes were identified as major sources of VEGF. In summary, VEGF expression in the bone marrow is restricted to certain stages of differentiation and maturation of myeloid cells and correlates with the FAB category.

Cerivastatin and atorvastatin inhibit IL-3-dependent differentiation and IgE-mediated histamine release in human basophils and downmodulate expression of the basophil-activation antigen CD203c/E-NPP3.

Recent data suggest that the statins, apart from their lipid-lowering activity, exhibit profound anti-inflammatory effects. Basophils are major proinflammatory effector cells in diverse pathologic reactions. We have examined the in vitro effects of five different statins on primary human basophils, their progenitors, and the basophil cell line KU-812. Preincubation of blood basophils with cerivastatin or atorvastatin (0.1-100 microM) for 24 h reduced their capacity to release histamine on immunoglobulin E (IgE)-dependent stimulation in a dose-dependent manner. These statins also inhibited IgE-dependent up-regulation of the basophil-activation antigen CD203c. Moreover, both statins suppressed interleukin-3-induced differentiation of basophils from their progenitors as well as (3)H-thymidine uptake in KU-812 cells. All inhibitory effects of cerivastatin and atorvastatin were reversed by mevalonic acid (200 microM). The other statins tested (lovastatin, simvastatin, pravastatin) did not show significant inhibitory effects on basophils. Together, these data identify cerivastatin and atorvastatin as novel inhibitors of growth and activation of human basophils.

C5a stimulates production of plasminogen activator inhibitor-1 in human mast cells and basophils.

We have recently shown that resting human mast cells (MCs) produce tissue-type plasminogen activator (t-PA) without simultaneously expressing plasminogen activator inhibitor 1 (PAI-1). In the present study we have identified the anaphylatoxin rhC5a as a potent inducer of PAI-1 expression in human MCs and basophils. In primary human skin MCs and primary blood basophils, exposure to rhC5a was followed by an increase from undetectable to significant levels of PAI-1. In addition, rhC5a induced a concentration- and time-dependent increase in PAI-1 antigen in the MC line HMC-1 and the basophil cell line KU-812 and increased the expression of PAI-1 mRNA in HMC-1. In conditioned media of HMC-1 treated with rhC5a, active PAI-1 could be detected. A simultaneous loss of t-PA activity in conditioned media from the same cells indicated that rhC5a-induced PAI-1 was capable of inhibiting the enzymatic activity of coproduced t-PA. Correspondingly, the levels of t-PA-PAI-1 complexes increased in rhC5a-treated cells. When HMC-1 cells were incubated with pertussis toxin or anti-C5a receptor antibodies, the effect of rhC5a on PAI-1 production was completely abolished. Treatment of C5a with plasmin resulted in loss of its ability to induce PAI-1 production in MCs. Considering the suggested role for MCs and components of the complement system in the development of cardiovascular diseases, we hypothesize that MCs, by producing t-PA in a resting state and by expressing PAI-1 when activated by C5a, might participate in the modulation of the balance between proteases and protease inhibitors regulating tissue injury and repair in such disease processes.