High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.

Interleukin 7 is produced by murine and human keratinocytes.

Interleukin 7 (IL-7) was originally identified as a growth factor for B cell progenitors, and subsequently has been shown to exert proliferative effects on T cell progenitors and mature peripheral T cells as well. Constitutive IL-7 mRNA expression so far had been demonstrated in bone marrow stromal cell lines, thymus, spleen, and among nonlymphoid tissues in liver and kidney. Here we show that both murine and human keratinocytes express IL-7 mRNA and release IL-7 protein in biologically relevant amounts. The physiological or pathological relevance of keratinocyte-derived IL-7 is presently unknown. Our finding that keratinocytes can produce IL-7 in concert with reports that IL-7 is a growth factor for in vivo primed antigen-specific T cells, as well as for T lymphoma cells suggests, however, that keratinocyte-derived IL-7 is important in the pathogenesis of inflammatory skin diseases and cutaneous T cell lymphoma.

Vitronectin shows complement-independent binding to isolated keratin filament aggregates.

Keratinocyte cell death, whether produced by skin disease or by physiologic apoptosis in normal skin, may result in formation of dermal keratin bodies, consisting mainly of keratin intermediate filament aggregates. Vitronectin, a multifunctional plasma and tissue glycoprotein, which inhibits the complement membrane attack complex and promotes cell attachment and spreading, is, like amyloid P component, associated with keratin bodies in vivo. To investigate a potential role for vitronectin in the removal of keratin bodies, we studied the interaction of vitronectin with keratin intermediate filaments in normal human skin and in Hep-2 cells, as well as with isolated keratin intermediate filament aggregates in vitro. Following pre-incubation of skin sections and Hep-2 cells with normal human serum (as a source of vitronectin), cytoplasmic staining of keratinocytes and of cytoskeletal filaments in Hep-2 cells was observed by immuno-fluorescence staining with polyclonal and monoclonal anti-vitronectin antibodies. Vitronectin binding to keratin intermediate filament aggregates extracted from normal human epidermis was demonstrated by immunofluorescence and by immunoblotting, and was not dependent on complement activation, because it occurred even when heat-inactivated human serum or C4-deficient serum was used as a source of vitronectin. Amyloid P component shows Ca++- dependent binding to keratin intermediate filament aggregates. does not involve amyloid P component because it occurred when binding of the latter protein was inhibited by EDTA buffer. Moreover, purified vitronectin also bound to keratin intermediate filament aggregates in immunofluorescence studies. Vitronectin binding to keratin intermediate filaments may play a role both in limiting complement-mediated tissue damage (because keratin bodies may activate complement) and in promoting removal of keratin bodies by fibroblasts and/or macrophages.

Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function.

Freshly isolated murine epidermal Langerhans cells (LC) are weak stimulators of resting T cells. Upon culture their phenotype changes, their stimulatory activity increases significantly, and they come to resemble lymphoid dendritic cells. Resident murine LC, therefore, might represent a reservoir of immature dendritic cells. We have now used enzyme cytochemistry, a panel of some 80 monoclonal antibodies, and immunofluorescence microscopy or two-color flow cytometry, as well as transmission electron microscopy, to analyse the phenotype and morphology of human LC before and after 2-4 d of bulk epidermal cell culture. In addition, LC were enriched from bulk epidermal cell cultures, and their stimulatory capacity was tested in the allogeneic mixed leukocyte reaction and the oxidative mitogenesis assay. Cultured human LC resembled human lymphoid dendritic cells in morphology, phenotype, and function. Specifically, LC became non-adherent upon culture and developed sheet-like processes (so-called "veils"), decreased their surface ATP/ADP'ase activity, and lost nonspecific esterase activity. As in the mouse, surface expression of MHC class I and II antigens increased significantly, and FcII receptors were significantly reduced. Markers that are expressed by dendritic cells (like CD40) appeared on LC following culture. Cultured human LC were potent T-cell stimulators. Our findings support the view that resident human LC, like murine LC, represent immature precursors of lymphoid dendritic cells in skin-draining lymph nodes.

Phagocytosis of keratin filament aggregates following opsonization with IgG-anti-keratin filament autoantibodies.

IgG-anti-keratin intermediate filament autoantibodies occur in low titers in all normal human sera. These same antibodies are present in high titers in the sera of patients who have diseases in which cells containing keratin intermediate filaments (KIF) have been damaged, such as systemic lupus erythematosus, graft-versus-host disease, and cutaneous tumors. Since some human autoantibodies are thought to function in part as opsonins promoting the removal of insoluble cellular proteins after tissue injury, we investigated the influence of IgG-anti-KIF autoantibodies on the phagocytosis of insoluble KIF aggregates by human monocytes and polymorphonuclear neutrophils (PMN). Keratin intermediate filaments assembled in vitro were reconstituted into dense spherical KIF aggregates 0.3-2.5 microns in diameter by dialysis against phosphate-buffered saline. Immunoelectron microscopy revealed that, as expected, human IgG-anti-KIF autoantibodies bound to the KIF aggregates. Human monocytes or PMN were incubated either with nonopsonized KIF aggregates or with KIF aggregates that had been reacted with IgG-anti-KIF autoantibodies. The uptake of KIF aggregates was visualized by indirect immunofluorescence, immunoperoxidase staining, and immunoelectron microscopy. Monocytes rapidly and efficiently bound and phagocytosed KIF aggregates that had been coated with IgG-anti-KIF autoantibodies. Nonopsonized KIF aggregates, in contrast, were taken up much less efficiently. Differences were most marked at 4 degrees C for 60 min with phagocytosis of opsonized KIF aggregates by 23 +/- 8% of monocytes in contrast to phagocytosis by only 0.2 +/- 3% monocytes when nonopsonized KIF aggregates were used. Similar results occurred at 37 degrees C for 5 min with phagocytosis by 38 +/- 28% vs 1.8 +/- 0.4% of monocytes of opsonized and nonopsonized KIF aggregates, respectively. A high percentage of PMN also phagocytosed opsonized KIF aggregates, whereas nonopsonized KIF aggregates were ingested less avidly. These data indicate that the opsonization of extracellular KIF aggregates by IgG-anti-KIF autoantibodies plays an important role in promoting the phagocytosis of KIF aggregates. The subsequent phagocytosis represents a rapid and very effective mechanism for the removal of insoluble KIF following keratinocyte cell death.

Apoptotic keratin bodies as autoantigen causing the production of IgM-anti-keratin intermediate filament autoantibodies.

The presence of numerous keratin bodies in the upper dermis is a characteristic finding in skin lesions of patients with various dermatoses such as cutaneous graft-versus-host disease, lichen planus, or chronic discoid lupus erythematosus. These keratin bodies are generated by apoptotic keratinocyte death, consist largely of keratin intermediate filaments (KIF), and are constantly covered with immunoglobulins, mainly IgM. Apoptosis is also thought to occur under physiologic conditions in the skin as it does in other organs, but keratin bodies are not frequently reported as being found in nonlesional skin. In order to assess the frequency of keratin bodies in normal skin, we examined serial sections of 10 normal human skin specimens and 5 dermal sheets prepared from normal human skin for the presence of keratin bodies. They were visualized by direct immunofluorescence using a fluorescein isothiocyanate (FITC) rabbit antihuman IgM conjugate. In addition the KIF origin of keratin bodies was demonstrated by a double-staining immunofluorescence procedure using a FITC-conjugated rabbit antihuman IgM followed by a mouse monoclonal antibody against keratin and a sheep antimouse immunoglobulin conjugated with Texas Red. One specimen was also examined for keratin bodies at the ultrastructural level. In serial sections, all 10 normal human skin specimens had numerous keratin bodies as assessed by visualization of globular IgM deposits. Evaluated on dermal sheets, the number of keratin bodies ranged from 39-262 per mm2. Nearly all keratin bodies also stained with the antikeratin antibodies. Ultrastructurally the remarkable number of keratin bodies, which consist of filaments measuring approximately 10 nm in diameter or of more granular material, in normal human skin was confirmed. In order to investigate the capacity of KIF material in keratin bodies to function as autoantigen, we examined the sera of the 10 skin donors and, in addition, of 30 normal healthy individuals and 10 patients with rheumatoid arthritis for the occurrence and specificity of IgM-anti-KIF autoantibodies by an enzyme-linked immunosorbent assay and by immunoblot. IgM-anti-KIF autoantibodies were found in all 50 test sera. In the majority of the sera the specificity of these autoantibodies included the 51 kD and the 58 kD KIF protein, which are constituents of KIF in keratin bodies and basal keratinocytes. Quantitatively, the antibody activity of the IgM-anti-KIF autoantibodies varied from serum to serum, being highest in the sera of patients with rheumatoid arthritis.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro complement binding in human skin cells with altered differentiation.

Exposure of cytoskeletal intermediate-sized filaments (ISF) of various cell populations in normal human skin to normal human serum (NHS) results in the deposition of C3 upon these structures; this phenomenon most likely occurs antibody-independently, is initiated by Clq binding to ISF, and is followed by the activation of the classical complement pathway. In the present study we investigated the cytoplasmic C3-binding properties of skin cells undergoing altered differentiation. Incubation of cryostat skin sections of dermal melanocytic nevi with NHS and, subsequently, with fluorescein isothiocyanate-conjugated rabbit antihuman C3 resulted in a bright cytoplasmic staining of the vast majority of nevus cells. Immunoelectron microscopic studies demonstrated that ISF within nevus cells represented the only cytoplasmic C3-binding structures. In contrast, ISF within melanoma cells, basal cell carcinoma cells, and keratinocytes constituting psoriatic lesions lacked C3-binding properties. We propose that changes in structure and subunit protein composition of ISF in certain cells undergoing altered differentiation results in a decrease or loss of their C3-binding capacity.

Deep penetrating dermatofibroma versus dermatofibrosarcoma protuberans. A clinicopathologic comparison.

A study of the clinical, histological, and immunohistochemical features of 20 cases of deep penetrating dermatofibroma (DPDF) and eight cases with 14 specimens (eight primary, one reexcision, five secondary tumors) of dermatofibrosarcoma protuberans (DFSP) showed distinct entities. Clinically, DPDF usually appeared as a nodule (approximately 2 cm) of the (lower) limbs, whereas DFSP affected the trunk (shoulder) with irregularly arranged plaques or nodules (> 5 cm). Histologically, DPDF showed a regular silhouette with a smooth, nodular (four of 20) or scalloped (16 of 20) lower margin and variable sclerosis (nine of 20); DFSP, irregularly infiltrated fatty tissue in a lacelike/honeycomb (eight of 14), multilayered (three of 14), or mixed pattern (three of 14), but without sclerosis. Immunohistochemically, DPDF was mostly negative with QBEnd 10 (CD34; 18 of 20) but positive for factor XIIIa (17 of 20), actin (HHF35; 10 of 20), and metallothionein (MT; 12 of 20). DFSP was positive for CD34 (13 of 14), yet with some sparing of central tumor parts, highly cellular tumor nodules, and myxoid areas; factor XIIIa and MT were consistently negative, as was HHF35 in 11 of 14 cases. In a multivariate analysis of histologic and immunohistochemical criteria, the combination of sclerosis and labeling with MT was most valid (p = 0.0001) for diagnosis: all DPDF showed either labeling with MT in "early" (metabolically active) lesions or sclerosis in "late" lesions, not present in DFSP.

Leptin receptor in human term placenta: in situ hybridization and immunohistochemical localization.

In the present study, we investigated the expression and localization of leptin receptors in human term placentae. On human term placenta tissue slices, digoxigenin-UTP labelled RNA-probe detected the long form of the leptin receptor ObR(L)mRNA in syncytiotrophoblasts of the villi, whereas the haematological subtype of the leptin receptor ObR/B219.1 was detected in blood cells of the intervillous space and fetal vessels. Immunohistochemistry, with two polyclonal antibodies to the N-terminus recognizing ObR(L)and ObR(S)of the leptin receptors and one to the C-terminus recognizing the long form of the leptin receptor ObR(L), localized leptin receptor protein at the apical membrane of the syncytiotrophoblasts. Our results show that the long form of the leptin receptor ObR(L)is expressed in human term placentae. We localized the long form of leptin receptor mRNA to the cytoplasm of syncytiotrophoblasts and leptin receptor proteins in human term placentae to the apical membrane of syncytiotrophoblasts. We conclude that in term placentae, leptin could mediate a growth promoting effect in the fetoplacental unit through the long form of the leptin receptor localized in the syncytiotrophoblasts. In contrast, the haematological subtype of the leptin receptor is not expressed in placental cells, but solely by blood cells in the intervillous space and fetal vessels.

Lipoprotein profile and cholesteryl ester transfer protein in neonates.

Undernourishment in utero appears to be associated with persisting changes in the metabolic, endocrine, and immune functions. In this study, we determined the influence of birth weight on the lipoprotein profile and cholesteryl ester transfer protein (CETP), which promotes a proatherogenic lipoprotein profile in plasma by determining the chemical, physical, and biologic properties of the respective lipoprotein particles. Triglyceride (TG) concentrations were highest and high-density lipoprotein (HDL)(2)-cholesterol levels were lowest in small for gestational age (SGA) neonates. CETP-mass was determined by enzyme-linked immunosorbent assay (ELISA) and CETP-activity by using exogenous lipoproteins. Cholesteryl ester transfer was determined as transfer of radiolabeled cholesteryl esters (CE) from HDL to apolipoprotein B-containing lipoproteins. CETP mass was lowest and cholesteryl ester transfer was highest in SGA neonates. CETP-activity did not differ among the neonates. Our results suggest that increased and decreased nourishment in utero affects the lipoprotein profile and CETP in neonates. High TG and low HDL(2) levels in SGA neonates might result from increased cholesteryl ester transfer and, may in part, explain the increased risk of coronary heart disease (CHD) of small for gestational age neonates in later life.

Enzyme-linked immunosorbent assay for detection of antibodies to the unmodified beta-lactam ring.

Dose- and pH- dependent carbodiimide-mediated coupling of Penicillin-G to polystyrene microtiter-plates that leaves the beta-lactam ring unchanged is described. A new ELISA method was developed using Penicillin-G coated plates. The binding of 3 different monoclonal antibodies as well as human IgG antibodies of the IgG1 and IgG3 subclasses is demonstrated, whereas IgG2, IgG4 and IgE antibodies did not bind. Thus, covalently coupled Penicillin-G can be used to study the immune-response to the unchanged beta-lactam ring in patients receiving penicillin therapy. The new method is complementary to hitherto described techniques, which generally only allow detection of antibodies binding to penicilloyl-groups.

Molecular characterization of rabbit scavenger receptor class B types I and II: portal to central vein gradient of expression in the liver.

To further elucidate the role of scavenger receptor class B type I (SR-BI) in reverse cholesterol transport and in atherogenesis, we performed studies in the rabbit, an animal model displaying a lipoprotein profile similar to that of human, expressing cholesteryl ester transfer protein in plasma and having been demonstrated to be susceptible to atherosclerosis. In this report, we describe for the first time the isolation and characterization of rabbit cDNA fragments encoding SR-BI and scavenger receptor class B type II (SR-BII). Development of an isoform-specific Taqman Real Time PCR system and generation of isoform-specific polyclonal antibodies allowed us to measure SR-BI and SR-BII expression in various rabbit organs on mRNA and protein levels, respectively. We found the highest expression of SR-BI in adrenal gland, liver, and proximal intestine; lesser expression was found in appendix and spleen. Immunohistochemical staining of frozen sections showed SR-BI expression in the cortex but not in the medulla of adrenal gland. An increasing portal to central vein gradient of expression was found within the hepatic lobule. As shown in this report, identification and characterization of SR-BI expression in the rabbit affords a powerful tool to elucidate the role of SR-BI in cholesterol homeostasis and atherogenesis in human.

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.