Serum of patients with Behçet's disease induces classical (pro-inflammatory) activation of human macrophages in vitro.

BACKGROUND: Although several immunological abnormalities have been demonstrated in Behçet's disease (BD), the exact mechanism of the inflammatory changes occurring is still unknown. Antigen-presenting cells, such as mononuclear phagocytes, play a major role in the regulation of immune-mediated as well as of non-specific inflammation. OBJECTIVE: To investigate the serum activity of patients with BD on antigen and chemokine expression of human macrophages in vitro. METHODS: Serum of 15 patients (8 women, 7 men; mean age 33 +/- 10 years) with BD was incubated with cultured macrophages isolated from peripheral blood of healthy volunteers. Macrophages maintained in patients' serum, fetal calf serum with/without dexamethasone and interleukin (IL)-4 or gamma-interferon were investigated for alternative macrophage-activation-associated CC-chemokine 1 (AMAC-1) and IL-8 mRNA expression by Northern blotting. In addition, cytocentrifuge macrophage preparations were stained with monoclonal antibodies against proteins indicating alternative (anti-inflammatory) macrophage activation, such as MS-1 high-molecular-weight protein (MS-1-HMWP), RM3/1 antigen (CD163) and 25F9, as well as classical (pro-inflammatory) macrophage activation, such as CD11c, class I receptor binding the Fc part of IgG (FcgammaRI: CD64) and class III receptor binding the Fc part of IgG (FcgammaRIII: CD16). RESULTS: Macrophages treated with patients' serum showed neither AMAC-1 expression nor staining with monoclonal antibodies for MS-1-HMWP, CD163 or 25F9. Concomitant treatment with IL-4/dexamethasone up-regulated significantly the expression of CD163. In contrast, IL-8 mRNA expression and staining for CD11c and CD64 were strongly positive after treatment with serum of patients with BD. CD64 positivity and IL-8 mRNA expression were more prominent in patients with active BD than in patients with inactive disease. CONCLUSION: Taken together, our results indicate that serum of patients with BD induces classical (pro-inflammatory) activation of human peripheral blood macrophages in vitro. Our findings suggest that serum factor(s) may be responsible for inflammatory changes in BD.

Pseudoexons and regulatory elements in the genomic sequence of the beta-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1.

Recently, the authors reported the cloning of a novel human CC chemokine of alternatively activated macrophages (AMAC-1), whose expression is induced by Th2-associated cytokines such as interleukin 4 (IL-4), IL-13 and IL-10; vice versa, AMAC-1 expression is inhibited by Th1-associated cytokines such as interferon gamma (IFN-gamma). In order to study the genomic organization and transcriptional regulation of the AMAC-1 gene, genomic clones were isolated by screening a human lambda genomic library. Sequencing of a clone with a 1.7-kb insert gave a partial genomic sequence for the AMAC-1 gene. The complete AMAC-1 genomic sequence was obtained by bioinformational methods and the whole region spanning the AMAC-1 gene was verified by PCR amplification of subfragments and sequencing. The AMAC-1 gene consists of three exons. Whereas exons 2 and 3 were separated by a small intron of 411 bp, exon 1 and exon 2 were separated by 6 kb of non-translated genomic sequence containing two pseudoexons that are not expressed although they feature intact exon/intron boundaries and complete open reading frames. In order to allow a detailed analysis, a 2.7-kb fragment containing the promoter region and the first exon of AMAC-1 gene was cloned into a reporter gene construct. In the AMAC-1 promoter, two possible transcription start points were identified. In addition, several putative regulatory sequences for IL-4- and IFN-gamma-dependent transcriptional pathways were found including STAT6 and STAT1 binding sites as well as several AP-1 and C/EBP elements. Interestingly, a combined STAT6/STAT1 binding element is located in the direct vicinity of the first putative transcription start point. Competitive binding of IL-4-induced STAT6 versus IFN-gamma-induced STAT1 to this site may explain the antagonistic effects these cytokines exert on AMAC-1 expression.

Alternative versus classical activation of macrophages.

In parallel to the Th1/Th2 paradigm, antigen-presenting cells (APC) are divided into classically activated APC (dendritic cells/effector macrophages) and alternatively activated APC (IL-4-induced, alternatively activated macrophages/IL-10-induced, immature dendritic cells). Alternatively activated APC share a special molecular repertoire including receptors of innate immunity with broad specificity for foreign antigen and anti-inflammatory cytokines such as IL-1Ra and alternative macrophage activation-associated CC-chemokine-1. Alternatively activated APC mediated tolerance and downregulated inflammation. Abuse of alternatively activated APC in support of infectious susceptibility or tumor immune escape is counteracted by the classical pathway. Thus, classically and alternatively activated APC secure the balance between proinflammatory and anti-inflammatory immune reactions.

Balanced macrophage activation hypothesis: a biological model for development of drugs targeted at macrophage functional states.

Macrophages play a central role in the immune response and are major targets for chronic infection with viruses such as HIV. Recent studies on macrophage differentiation have shown the existence of classical activation and the counter-balancing anti-inflammatory alternative activation states. In the 'balanced macrophage activation hypothesis' we propose that macrophage activation is a cyclic process that balances these two states to achieve proper immunologic function. Dysregulation of this cycle would, therefore, be associated with various forms of chronic disease. This model has been utilized in the drug development of WF10, a novel macrophage-targeted drug, currently in advanced clinical testing for the treatment of HIV disease.

Langerhans cells do not express alternative macrophage activation-associated CC chemokine (AMAC)-1.

We have cloned a novel human CC chemokine, alternative macrophage activation-associated CC chemokine (AMAC)-1 that is highly homologous to macrophage inflammatory protein (MIP)-1alpha. In contrast to MIP-1alpha, AMAC-1 is induced in macrophages by Th2-associated cytokines IL4, IL13, and IL10 in vitro; in addition, AMAC-1 is expressed by Th1-suppressive alveolar macrophages in vivo. Surprisingly, however, AMAC-1 is also expressed by GM-CSF-induced, in vitro monocyte-derived dendritic cells when treated by IL4. Here, we present a detailed analysis of AMAC-1 expression in monocyte-derived dendritic cells in vitro and show that the prime dendritic cells in vivo, i.e. epidermal Langerhans cells, do not express AMAC-1 mRNA. In conclusion, AMAC-1 is a novel CC chemokine whose Th2-associated expression pattern in alternatively activated suppressor macrophages in vivo and in vitro and its absence from epidermal Langerhans cells in vivo suggest that it may be involved in inhibition of Th1 reactions and in tolerance induction.

Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern.

We have cloned a novel human CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. The isolated cDNA clone (803 bp) shows a single open reading frame of 267-bp coding for 89 amino acid residues; mature AMAC-1 protein is predicted to consist of 69 amino acids with a m.w. of 7855. Sequence alignment and 3D-modeling show the typical structural characteristics of CC-chemokines with special features in the receptor-activating domain. AMAC-1 is most closely related to MIP-1 alpha with a cDNA and protein sequence homology of 55% and 59%, respectively. However, the expression pattern of AMAC-1 is directly opposite to that of MIP-1 alpha. While MIP-1 alpha is induced by classical macrophage mediators such as LPS and is inhibited by IL-4 and glucocorticoids, AMAC-1 is specifically induced in macrophages by alternative macrophage mediators such as IL-4, IL-13, and IL-10. Expression of AMAC-1 is inhibited by IFN-gamma while glucocorticoids exert a slightly positive synergistic effect in combination with IL-4. Peripheral blood monocytes do not express AMAC-1; time course experiments show that monocyte-to-macrophage differentiation is a prerequisite for AMAC-1 expression. Expression of AMAC-1 by granulocyte-macrophage CSF/IL-4-induced, monocyte-derived dendritic cells is complex; in mature adherent dendritic cells, however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1 is expressed by alveolar macrophages from healthy persons, smokers, and asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose expression is induced in alternatively activated macrophages by Th2-associated cytokines; thus, AMAC-1 may be involved in the APC-dependent T cell development in inflammatory and immune reactions.

Evidence of heterogeneity and quantitative differences of the type 1 5alpha-reductase expression in cultured human skin cells--evidence of its presence in melanocytes.

Steroid 5alpha-reductase is of crucial importance in androgen physiology because it catalyzes the conversion of testosterone into the more potent 5alpha-dihydrotestosterone in androgen-regulated target tissues. The enzyme occurs in two isoforms, whereby type 1 isozyme exists mainly in the skin and type 2 in the prostate. By using human cell cultures, we examined cutaneous expression and subcellular localization of type 1 5alpha-reductase in vitro. In immunocytochemistry, type 1 5alpha-reductase was detected in the cytoplasm of cultured human sebocytes, keratinocytes, fibroblasts, dermal microvascular endothelial cells, hair dermal papilla cells, and melanocytes. In western blot studies, two closely lying bands of 21-27 kDa were detected, possibly indicating heterogeneity of the type 1 5alpha-reductase in all the cell types tested, with the exception of beard dermal papilla cells. Northern blot studies revealed most abundant type 1 mRNA in neonatal foreskin keratinocytes, followed by adult facial sebocytes. Occipital hair dermal papilla cells presented higher levels of type 1 5alpha-reductase mRNA than those of beard. These findings were confirmed by semiquantitative reverse transcriptase polymerase chain reaction coupled with high performance liquid chromatography analysis. Taken together, it seems likely that in cultured human skin cells there exist (i) heterogeneity of type 1 5alpha-reductase protein and (ii) quantitative differences in its transcriptional and translational expression levels.

Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle.

Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.

Differences in angiogenic potential of classically vs alternatively activated macrophages.

Macrophages (M phi) are important for angiogenesis during inflammation, wound repair, and tumor growth. However, well-characterized M phi subsets such as IFN-gamma-induced, classically activated (ca) M phi or IL-4/glucocorticoid-induced, alternatively activated (aa) M phi have not been thoroughly examined for a positive or negative association with angiogenesis. While caM phi populate early inflammatory reactions and high-turnover granulomas, aaM phi occur in healing wounds and chronic inflammation. In contrast to caM phi-dominated lesions, aaM phi-rich lesions are highly vascularized. In order to determine their angiogenic potential in vitro, these M phi subsets as well as unstimulated control macrophages (coM phi) were analyzed by RT-PCR for mRNA expression of 10 angiogenic factors after 3 and 6 days of culture. Early during activation, caM phi and coM phi expressed equal levels of 8 of 10 angiogenic factors (PDGF-A, MK, TNF-alpha, TGF-beta 1, PDGF-B, HGF, TGF-alpha, IGF-1), while aaM phi showed expression of only 4 of these factors (TGF-beta 1, PDGF-B, HGF, GF-1). After maturation, TGF-alpha and IGF-1 showed a shift in mRNA expression from caM phi to aaM phi resulting in a considerably enhanced expression of these factors in day-6 aaM phi as compared to day-6 caM phi and coM phi while PDGF-A, MK, and TNF-alpha remained suppressed in day 6 aaM phi. In all M phi subsets including controls, mRNA expression of aFGF and bFGF was minimal or absent while TGFG-beta 1, HGF, and ODGF-B were constitutively expressed. In order to functionally integrate angiogenic factor mRNA expression profiles, mitogenic activity of M phi subsets towards microvascular endothelium was assessed by cocultivation. Coculture experiments revealed that endothelial proliferation induced by aaM phi was 3.0-3.5x higher than induced by caM phi. In conclusion, mature aaM phi are well equipped to play an important role in protracted M phi-associated angiogenic processes. Presumably due to expression of predominantly angio-inhibitory cytokines such as TNF-alpha by caM phi but much less by aaM phi, caM phi exhibit only a low angiogenic potential in vitro and in vivo despite considerable expression of angiogenic factor mRNA.

1alpha,25-dihydroxyvitamin D3 induces sphingomyelin hydrolysis in HaCaT cells via tumor necrosis factor alpha.

Treatment of the human keratinocyte cell line HaCaT with 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in the hydrolysis of sphingomyelin with peak elevations of ceramide levels after 2-3 h (Geilen, C. C., Bektas, M., Wieder, Th., and Orfanos, C. E. (1996) FEBS Lett. 378, 88-92). In the present paper, the mechanism underlying the effect of 1,25-(OH)2D3 on sphingomyelin hydrolysis was investigated. Using the cell culture supernatant of HaCaT cells treated with 1,25-(OH)2D3 for 2 h, it was possible to induce sphingomyelin hydrolysis as early as 30-60 min after addition to resting cells. Several lines of experimental evidence indicated that tumor necrosis factor alpha (TNFalpha) mediates sphingomyelin hydrolysis after 1,25-(OH)2D3 treatment: (i) 1,25-(OH)2D3 stimulated TNFalpha mRNA expression after 1 h, (ii) newly synthesized TNFalpha occurred 2 h after 1,25-(OH)2D3 treatment, (iii) indirect activation of sphingomyelin hydrolysis by the supernatant of 1, 25-(OH)2D3-treated HaCaT cells was abolished by preincubation of the supernatant with antibodies directed against TNFalpha, and (iv) preincubation of HaCaT cells with neutralizing antibodies directed against the 55-kDa receptor of TNFalpha blocked the ability of 1, 25-(OH)2D3 to induce sphingomyelin hydrolysis in HaCaT cells. These data demonstrate that 1,25-(OH)2D3 activated sphingomyelin hydrolysis by an autocrine mechanism via TNFalpha expression. Furthermore, this indirect mode of action may serve as an explanation for the delayed induction of sphingomyelin hydrolysis by vitamin D3.

L-ascorbic acid inhibits UVA-induced lipid peroxidation and secretion of IL-1alpha and IL-6 in cultured human keratinocytes in vitro.

We investigated the antioxidative effect of L-ascorbic acid on lipid peroxidation and on secretion and mRNA expression of IL-1alpha and IL-6 after UVA irradiation (20 J/cm2) in cultured human keratinocytes. Lipid peroxidation was measured by (i) high performance liquid chromatography with UV detection of malondialdehyde (MDA) at 256 nm and (ii) spectrometric measurement of thiobarbituric acid-reactive substances (TBARS). To evaluate UV-induced cytotoxicity, we assessed cell membrane damage by measuring lactate dehydrogenase (LDH) release. UVA-induced lipid peroxidation in cultured human keratinocytes was inhibited by ascorbic acid in a concentration-dependent manner: MDA protein equivalent was reduced by 47% (10(-6)), compared to keratinocytes not exposed to L-ascorbic acid (p < 0.05), and the TBARS showed a concentration-dependent decrease of 49% (10(-6) M) in L-ascorbic acid-supplemented cultures compared to controls (p < 0.05). LDH release was decreased by 45% in L-ascorbic acid-supplemented keratinocyte cultures, indicating protection against cell death (p < 0.05). L-Ascorbic acid was able to downregulate IL-1alpha mRNA expression in both UVA-irradiated and nonirradiated cells; however, IL-6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L-ascorbic acid. These findings indicate a major cell-protective effect of L-ascorbic acid on UVA-induced lipid peroxidation and the secretion of pro-inflammatory cytokines by UVA-irradiated human keratinocytes.

Alternatively activated macrophages actively inhibit proliferation of peripheral blood lymphocytes and CD4+ T cells in vitro.

We compared the immunological functions of interferon-gamma (IFN-gamma)-induced, classically activated macrophages (caM phi) and of interleukin-4 (IL-4)- and glucocorticoid-induced, alternatively activated macrophages (aaM phi) in a human co-culture system in vitro. Proliferation of peripheral blood leucocytes (PBL) or CD4+ T cells mediated by optimal doses of phytohaemagglutinin (PHA) or concanavalin A (Con A) was only marginally influenced by caM phi, but was strongly inhibited by aaM phi. The degree of lymphocyte proliferation sustained in the presence of caM phi was gradually reduced in a dose-dependent fashion by the addition of aaM phi. Flow cytometric analysis revealed that expression of costimulatory molecules such as CD11a, CD40, CD54, CD58, CD80 and CD86 did not vary significantly between caM phi and aaM phi and was low for CD58, CD80 and CD86. As shown by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, IL-10 was expressed in caM phi, aaM phi and control macrophages; the level of expression of IL-10 was slightly enhanced in aaM phi. Neither neutralizing anti-IL-10 antibodies, indomethacin nor NG-monomethyl-L-arginine (NMMLA) was able to reverse aaM phi-mediated inhibition of lymphocyte proliferation. Of several agents interfering with various second messenger pathways, cAMP and the Ca(2+)-ionophore A23187 inhibited differentiation of cultured human monocytes into phenotypically mature aaM phi expressing MS-1 high molecular weight protein (MS-1-HMWP) and RM 3/1 antigen, and prevented the suppressive action of aaM phi on lymphocyte proliferation. In conclusion, these results who that aaM phi actively inhibit mitogen-mediated proliferation of PBL and CD4+ T cells independently of the expression of costimulatory molecules and of IL-10, NO or prostaglandin synthesis, and that inhibition of phenotypic differentiation of aaM phi is paralleled by a lack of functional maturation. Thus, fully matured aaM phi may be functional in down-regulating CD4+ T-cell-mediated immune reactions by an as yet unknown mechanism.

Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.

Immunological mechanisms play an important role in the pathogenesis of psoriasis. Lesional psoriatic skin-derived T-cell clones have been shown to stimulate keratinocyte proliferation and to predominantly express a T-helper type 1 cytokine pattern. However, T-helper type 2-like cytokines have also been identified in some psoriatic T-cell clones. In parallel to the T-helper type 1/type 2 dichotomy, a distinction between interferon-gamma-induced (classically activated) macrophages and interleukin-4/glucocorticoid-induced (alternatively activated) macrophages has been put forward as a conceptual framework for a better understanding of immunopathological processes. In the present study, the phenotype of mononuclear phagocytes in psoriatic skin lesions (n = 21), allergic contact dermatitis (n = 4) and normal skin (n = 2) was investigated using a panel of monoclonal antibodies (mAb) against monocytes/macrophages and dendritic cells (mAb MS-1, RM 3/1, and 25F9 against subsets of in vitro alternatively activated macrophages, and mAb against myeloid antigens CD1a, CD11b, CD11c, CD34, CD36, and CD68). With regard to mononuclear phagocytes, psoriatic skin was found to be compartmentalized into epidermis, subepidermal space, and upper and lower dermis. RM 3/1++ +, MS-1+/-, 25F9- dendritic macrophages previously classified as type II alternatively activated macrophages were the dominant dermal macrophage population in psoriatic skin, while intraepidermal and epithelium-lining macrophages expressed a different, presumably classically activated, macrophage phenotype (RM 3/1-, MS-1-, 25F9-, CD68+2, CD11b+2). In allergic contact dermatitis, a classical T-helper type 1 disease, RM 3/1++ + macrophages were less prominent. Since MS-1 high molecular weight protein is much more sensitive to interferon-gamma-induced suppression than RM 3/1 antigen, a predominance of T-helper type 1 cytokines in psoriasis could explain why dermal dendritic macrophages do not express the fully induced MS-1++ +, RM 3/1++ +, 25F9+/- phenotype of type I alternatively activated macrophages.

The mononuclear phagocyte-dendritic cell dichotomy: myths, facts, and a revised concept.

Since Aschoff's reticuloendothelial system was abandoned a few decades ago, classification and characterization of the mononuclear phagocyte and dendritic cell systems have evolved separately or even in competition with one another. New information has now become available indicating that monocytes/macrophages and dendritic cells have a common origin in the bone marrow, and may even transdifferentiate. Morphological and functional distinctions-although valid under certain conditions-have been blurred by revelation of the versatility of monocytes/macrophages and dendritic cells in response to different contextual needs in inflammation and immunity. Monocytes/macrophages and dendritic cells share a sentinel, receptor/effector, and presentation mode, and may either activate or silence specific immune reactions. In keeping with the view of monocytes/macrophages and dendritic cells as interactive sentinels, we suggest that the mono-nuclear phagocyte and dendritic cell systems be replaced by the custocyte system (custos, Lat = sentinel, guard) as a unifying concept. Within the custocyte system, we recognize type I, type II, and type III custocytes. Type I and II custocytes exhibit predominance of presentation or effector/presenter interdependency, respectively, while type III custocytes are bipolar, passing through type I- and type II-like phases during their development and in inflammatory responses. The custocyte system brings into view monocytes/macrophages and dendritic cells as dynamic players in immunity and inflammation with a high degree of derivational, phenotypic, functional, and molecular plasticity.

Dissection of macrophage differentiation pathways in cutaneous macrophage disorders and in vitro.

Macrophages play important roles in immunity and inflammation, and in allergic, granulomatous and neoplastic diseases. Here, we present the indepth results of an ongoing study of macrophage differentiation pathways in cutaneous macrophage disorders and in vitro. Up to now, a total of 40 cases of cutaneous macrophage disorders (histiocytoses and granulomas) and related diseases were examined using a panel of monoclonal and polyclonal antibodies to macrophage differentiation antigens (mAb MS-1, mAb alpha CD1a, mAb alpha CD34, mAb RM 3/1, mAb alpha CD11c, mAb alpha CD36, mAb MAC 387, mAb 27E10, polyclonal antibodies alpha MRP-8 and -14, mAb alpha CD68, mAb 25F9, mAb DRC1-R4/23, and mAb 1F10). Of these, MS-1 high molecular weight protein, synthesized by non-continuous sinusoidal endothelial cells and highly dendritic perivascular macrophages in normal human organs, is the most specific macrophage differentiation marker. MS-1 high molecular weight protein is selectively expressed by cutaneous non-Langerhans cell histocytoses, and proves to be a valuable diagnostic tool for these diseases. MS-1 high molecular weight protein is not found in Langerhans cell histiocytosis cells, epithelioid cells in sarcoidosis, and palisading histiocytes in granuloma annulare. MS-1+ macrophages may be found intermingled in cellular type dermatofibroma and in foreign body granulomas; they differ from MS-1+ non-Langerhans cell histiocytosis cells by their highly dendritic morphology, and thus rather resemble the MS-1+ macrophages in normal skin. RM 3/1 antigen shows a similar, but broader expression pattern including non-Langerhans cell histiocytoses, xanthelasmata palpebrarum, foreign body granulomas, granuloma annulare, and cellular type dermatofibroma. Moreover, xanthelasmata palpebrarum paradigmatically represent a class of macrophage lesions with strong RM 3/1, but little MS-1 antigen expression. In sarcoidosis, RM 3/1+ macrophages are only found at the very periphery of epithelioid cell granulomas. In contrast, 25F9 antigen is strongly and consistently expressed in epithelioid cells of sarcoidosis, and in foreign body granulomas. In cultured human monocytes/macrophages, RM 3/1 antigen is expressed early on, while MS-1 high molecular weight protein and 25F9 antigen are late and very late macrophage differentiation antigens, respectively. Expression of RM 3/1 antigen and MS-1 high molecular weight protein is inducible by glucocorticoid and interleukin-4, and less so by interleukin-13 and interleukin-10, and combinations thereof, while 25F9 antigen seems to be less influenced by these agents. Interferon-gamma (and less so tumor necrosis factor-alpha) inhibit expression of all three antigens in cultured human monocytes/macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

T helper target cell DNA fragmentation through a CD4-positive T suppressor cell clone inducing specific unresponsiveness.

The CD4-positive bovine serum albumin (BSA)-specific Ts cell clone BVI/5 from a CBA/J mouse tolerized to low doses of BSA induces specific unresponsiveness in the T helper (Th) cell population. Tolerance induction can be measured in vitro in proliferation assays using specific Th cell clones or antigen-primed lymph node cells (LNC) and determined in vivo by the failure to produce hapten-specific antibodies. Using the BSA-specific Th cell clone 83/1 as a target one observes in addition 51Cr-release in a 16-hr long-term assay but finds no effect in a typical 6-hr T cell cytotoxicity test. BVI/5 Ts cells do not produce interleukin-2 but otherwise express a Th1 profile. The suppression of proliferation of 83/1 Th cells is partly due to interferon-gamma (IFN-gamma). But lysis of 83/1 Th cells as well as suppression of BSA-specific LNC proliferation needs direct cell contact between BVI/5 Ts cells and their targets. Cell lysis and suppression of LNC cannot be simulated by IFN-gamma, by the combination of IFN-gamma and TNF, or by BVI/5 supernatants. Thus mediators cannot account for specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from LNC and are probably not responsible for the induction of in vivo unresponsiveness. Instead the data show that BVI/5 Ts cells induce apoptosis-like DNA fragmentation in cloned BSA-specific 83/1 Th cells and in LNC from BSA-primed mice. Apoptosis can also be visualized as chromatin condensation in the LNC population. Macrophages have been excluded as targets. It can further be demonstrated that BVI/5 Ts cells express perforin and granzyme A on activation. Thus they are equipped with the effector molecules for target cell destruction. We consider BVI/5 Ts cells to be representative of a regulatory T cell inducing specific unresponsiveness in peripheral lymphoid organs.

Characterization of idiotype-specific I-Ed-restricted T suppressor lymphocytes which confine immunoglobulin class expression to IgM in the anti-alpha (1- > 3) Dextran B 1355 S response of BALB/c mice.

The humoral immune response of conventionally raised BALB/c mice to the so-called "thymus independent" antigen alpha (1- > 3) Dextran B 1355 S (Dex) is predominantly of the IgM class. The response is further characterized by Igha allotype linkage and the dominance of the public idiotypes (Id) J558 and MOPC 104. In germfree raised BALB/c and in BALB/c nu/nu mice immunized with the same antigen an additional IgG response of the public Id is observed. Analysis of the regulation of the class expression reveals existence of specific Ts cells in euthymic mice which must have been activated pre- or perinatally by exposure to environmental bacterial antigens. They permit or enforce differentiation of Dex-specific B cells into B gamma memory cells without allowing further development into IgG producing plasma cells. An analogue of these splenic Ts cells has now been cloned and identified as an I-Ed restricted Id-specific T cell with exactly the properties ascribed above to the splenic Ts cells. This paper describes phenotypical and functional properties of the Ts cell clone 178-4. It evaluates this clone's role in controlling efficient anti-bacterial IgM-mediated immunity under conditions where a class switch to IgG antibody production is actively suppressed; possibly as a measure to avoid hazardous autoimmune reactions on the basis of crossreaction and antigenic mimicry between polysaccharide antigens.

Identification of a carcinoembryonic antigen gene family in the rat. Analysis of the N-terminal domains reveals immunoglobulin-like, hypervariable regions.

The existence of a carcinoembryonic antigen (CEA)-like gene family in rat has been demonstrated through isolation and sequencing of the N-terminal domain exons of presumably five discrete genes (rnCGM1-5). This finding will allow for the first time the study of functional and clinical aspects of the tumor marker CEA and related antigens in an animal model. Sequence comparison with the corresponding regions of members of the human CEA gene family revealed a relatively low similarity at the amino acid level, which indicates rapid divergence of the CEA gene family during evolution and explains the lack of cross-reactivity of rat CEA-like antigens with antibodies directed against human CEA. The N-terminal domains of the rat CEA-like proteins show structural similarity to immunoglobulin variable domains, including the presence of hypervariable regions, which points to a possible receptor function of the CEA family members. Although so far only one of the five rat CEA-like genes could be shown to be transcriptionally active, multiple mRNA species derived from other members of the rat CEA-like gene family have been found to be differentially expressed in rat placenta and liver.