N-Acetylcysteine downregulates vascular endothelial growth factor production by human keratinocytes in vitro

Abstract The present study was designed to evaluate the action of various antioxidants including N-acetylcysteine (NAC) and the flavonoids resveratrol and quercetin on the production of VEGF by human keratinocytes (HKC). NAC, resveratrol, and quercetin dose-dependently suppressed the incorporation of ³H-thymidine into HKC. Values of median inhibitory concentration for NAC, resveratrol, and quercetin were 10 mM, 55 μM, and 15 μM, respectively (P < 0.01). RT-PCR demonstrated VEGF 121 and VEGF 206 expression in all HKC samples. HKC showed baseline expression and a progressive gradual time-dependent increase in VEGF secretion (510 ± 75 pg/ml at 24 h), and EGF (2.5–100 ng/ml) enhanced the secretion of VEGF in a dose-dependent fashion. HKC were incubated with NAC (2.5–20 mM) for 2 h prior to the addition of EGF (5 ng/ml) or PMA (10 ng/ml), and a significant decrease (P < 0.01) was found after 24 h of incubation with 2.5 mM NAC. However, neither resveratrol nor quercetin reduced the synthesis of this cytokine. In summary we conclude that NAC and the flavonoid antioxidants resveratrol and quercetin inhibit HKC proliferation regardless of the stage of differentiation and that NAC significantly inhibits VEGF secretion in basal and EGF- or PMA-treated HKC. Received: 28 February 2000 / Revised: 21 July 2000 / Accepted: 11 October 2000     

Increased expression of CD208 (DC-LAMP) in epidermal keratinocytes of psoriatic lesions

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperproliferation and infiltration of inflammatory leukocytes. The aim of this study was to clarify the role of innate immunity involving dendritic cells (DC) and keratinocytes in psoriasis. We immunohistochemically examined the expression of DC markers such as CD1a, CD83, CD207 (Langerin), CD208 (DC-LAMP) and CD209 (DC-SIGN) in psoriatic skin and γ-interferon (IFN-γ)/12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated keratinocytes in vitro . CD208 was strongly expressed in basal and suprabasal layer keratinocytes in addition to DC in the perivascular lesions of the psoriatic dermis. Furthermore, the enhanced expression of CD208 in the perinuclear lesions of IFN-γ-/TPA-stimulated keratinocytes was observed in vitro . Because a defect of the granular layer in psoriatic lesions has been recognized, increased expression of lysosome-related CD208 in the basal and suprabasal keratinocytes of psoriatic lesions might represent aberrant epidermal differentiation. Additionally, these CD208-positive keratinocytes possessing putative antigen-processing activity might play a key role as antigen-presenting cells in psoriatic skin.     

Heat-stable antigen is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation

Abstract Heat-stable antigen (HSA), expressed by various antigen-presenting cells (APC), has been described as a costimulatory molecule for CD4⁺ T cells. Recently, we observed that HSA also serves as an important costimulatory molecule on epidermal Langerhans cells (LC). During these studies, low levels of HSA staining were also detected on normal murine keratinocytes (KC). To investigate whether HSA also is involved in T-cell activation by KC, normal murine KC or the spontaneously transformed KC cell-line PAM 212 were treated with PDB or PMA to induce HSA-expression. FACS analyses showed induction of HSA expression on normal murine KC, as well as PAM 212 cells. In functional assays PDB or PMA-treated normal or transformed KC were far more potent inducers of primary allogeneic T-cell responses than untreated KC. Addition of anti-HSA-specific mAb 20C9 specifically inhibited the costimulatory activity of KC, an effect that was even more pronounced when CTLA-4Ig was added to the cultures. Cleavage of HSA on KC surfaces by a phosphoinositol-specific phospholipase C (PI-PLC) also significantly inhibited the costimulatory capacity of KC for naive CD4⁺ T cells. In aggregate, our data indicate that expression of HSA on activated KC contributes to the capacity of these cells to induce proliferation of allogeneic T cells.     

Plasma concentrations of IFN-γ and TNF-α: in psoriatic patients before and after local treatment with dithranol ointment

IFN-γ and TNF-α plasma levels were measured before and after local treatment in 27 patients. Twenty healthy subjects served as controls. Plasma concentrations of IFN-γ and TNF-α were significantly higher before treatment (178.7 ± 11.9 pg/ml and 31.9 ± 11.6 pg/ml, respectively) compared to the control group (139.6 ± 7.86 pg/ml and 17.1 ± 7.7 pg/ml, respectively). After treatment IFN-γ levels were significantly decreased (151.3 ± 8.3 pg/ml) toward the control group values and TNF-α levels were observed even lower than in the controls (11.48 ± 6.8 pg/ml). No correlations were found between age, duration of psoriasis and plasma levels of cytokines. However, IFN-γ levels were related, although not significantly, to disease severity (evidenced by the PASI score). The data support the important proinflammatory role of IFN-γ and TNF-α in the clinical manifestation of psoriasis. © 1998 Elsevier Science B.V.     

Human HMC-1 mast cells exclusively express the FcγRII subtype of IgG receptor

Because of their localization at the interface of the internal and external environment mast cells play a crucial role in the immune response and in inflammatory reactions. Effects may be mediated not only by the high-affinity IgE receptor, but also by IgG receptors. Since in rodent mast cells signal transduction via the Fcγ receptor family has been shown, we analysed the expression of surface receptors for IgG on the human mast cell line HMC-1. It was shown by flow cytometric analysis that HMC-1 constitutively expressed the FcγRII/CD32 subtype whereas FcγRI/CD64 and FcγRIII/CD16 were not expressed. This exclusive expression of the FcγRII subtype of IgG receptor is similar to the expression pattern of basophils, although concerning cell surface molecules HMC-1 rather seem to resemble monocytes. In contrast to monocytes the expression profile on HMC-1 did not change upon stimulation with IL-4, TNFα, IFNγ, PMA or salbutamol. Moreover, the mast cell-activating cytokine SCF and the calcium ionophore A23187 did not modulate the FcγR profile in this study. To assess the importance of the exclusive FcγRII expression on HMC-1, we investigated whether the production of the cytokine TNFα is modulated via FcγRII activation or if an increase in intracellular calcium could be observed. No significant modulation of TNFα release or of intracellular free calcium after crosslinking of FcγRII by heat-aggregated IgG or by a second antibody was observed. It remains to be clarified whether this low-affinity subtype for the IgG receptor is involved in antigen-dependent sensitization of human tissue mast cells resulting in secretion of immunoregulatory cytokines. This mechanism may be important for disease states associated with circulating or tissue-bound immune complexes. Received: 26 February 1996     

A patient with plaque-stage mycosis fungoides has successfully been treated with long-term administration of IFN-γ and has been in complete remission for more than 6 years

Summary We report the successful treatment of a patient with plaque-stage mycosis fungoides with long-term and intravenous administration of recombinant human interferon-γ (IFN-γ) and discuss the possible mechanisms of this therapy.
A 55-year-old female patient had been resistant to existing treatments and had suffered repeated exacerbations over a 5-year period. Four weeks after initiation of 2 × 10⁶U/day of IFN-γ. a > 10% decrease in the affected surface area was noted. Twenty-two weeks after the administration of 228 × 10⁶U of IFN-γ, complete remission (CR) was obtained. The CR continued for 13 weeks, but this was followed by an exacerbation. The second CR was obtained after the IFN-γ dosage was increased to 16 × 10⁶U/week. The dosage was then gradually reduced by 2–4 × 10⁶U every 2 or 3 months. She was treated with a total dose of 2814 × 10⁶ of IFN-γ. She has been followed up for more than 6 years, and there has been no recurrence of mycotic skin lesions nor any visceral involvement. During therapy, no serious side-effects were noted.
Long-term administration of IFN-γ is useful for the treatment of patients with intractable mycosis fungoides. A gradual decrease in the dose of IFN-γ is important for maintaining remission.     

Enhanced allogeneic skin-graft survival using sCD95L, sCD152, interleukin-10 and transforming growth factor-β in combination, and comparison with ciclosporin

Background.  CD152 is a negative regulator of T-cell activation. The ligand of CD95, CD95L, induces apoptosis in CD95-expressing cells through activation of caspases. Interleukin (IL)-10 and transforming growth factor (TGF)-β have been implicated in suppressing immune response. Independent treatment with the four factors can induce immunotolerance and prolong the survival of grafts.
Aim.  To investigate whether joint use of murine (m)IL-10, human (h)TGF-β and our engineered soluble extracellular part of CD152 and CD95L (sCD152, sCD95L) has a synergistic effect on immunotolerance induction in allogeneic skin transplantation.
Methods.  Recipient mice were treated with sCD152, sCD95L, mIL-10 and hTGF-β, separately or combined. The survival time of skin allografts was observed and mixed lymphocyte reactions were performed to detect the reactivity of splenic cells from recipient mice compared with splenic cells from donors or from unrelated Institute of Cancer Research mice. The levels of cytokines such as IL-2, IL-4 and interferon (IFN)-γ, and antibodies specific for engineered sCD152 or sCD95L in serum were determined by ELISA.
Results.  Combined treatment with sCD152 and sCD95L synergistically prolonged the mean survival time (MST) of skin allografts, but the addition of mIL-10 or hTGF-β to these did not improve MST further.
Conclusion.  sCD152 and/or sCD95L regulate the differentiation of T-helper (Th) cells and induce the cytokine production shift of Th1-associated and Th2-associated cells. Both IFN-γ and IL-2 were negatively correlated and IL-4 was positively correlated with skin MST. Cytokines such as IFN-γ, IL-2 and IL-4 might function as indicators for predicting graft survival time.     

Infiltrating cells and related cytokines in lesional skin of patients with chronic idiopathic urticaria and positive autologous serum skin test

Abstract:  In approximately one-third of patients with chronic idiopathic urticaria (CIU), autoantibodies against the high-affinity IgE receptor and/or against IgE can be detected and a wheal-and-flare response can be provoked by the intradermal injection of autologous serum (ASST). In this study we aimed to further characterize the inflammatory response observed in the subgroup of CIU patients with positive ASST and serum-evoked histamine-release in vitro from basophils in comparison with unaffected skin and healthy donors. An immunohistochemical analysis of infiltrating cells (CD4, MPO, EG1, EG2, tryptase), cytokines (IL-4, IL-5, IFN-γ), chemokines and chemokine receptors (IL-8, CCR3, CXCR3), and adhesion molecules (ICAM-1, VCAM-1, ELAM-1) was performed on seven selected patients (four males and three females; median age: 45 years; range: 22–57) and five healthy donors. Cytokine evaluation was also performed in five psoriatic patients to obtain an additional control .
In spontaneous wheals we observed an increased number of CD4+ T lymphocytes when compared with the controls, and an increased number of neutrophils and eosinophils, whereas mast cells did not show a significant variation. A significant expression for IL-4 and IL-5 could only be observed in lesional skin, while IFN-γ showed a slight expression in the same site. Chemokine receptors CCR3 and CXCR3 did not show a defined polarized response in either lesional or unaffected skin. An increased expression of all cellular adhesion molecules (CAMs) studied was detected in spontaneous wheals. The lack of a significant difference in the expression of tryptase + mast cells, T lymphocytes, IL-8, CXCR3 and CCR3, a few CAMs between the lesional and unaffected skin of CIU patients suggests a wide immunological activation that involves not only lesional tissues, but possibly extends to the whole of the skin's immune system.     

α-MSH regulates interleukin-10 expression by human keratinocytes

Abstract Normal human keratinocytes (HKC) are able to synthesize α-MSH. Because the production of α-MSH by HKC is induced significantly by ultraviolet B radiation, the involvement of keratinocyte-derived α-MSH in UV-induced immunosuppression has been suggested. The induction of the antiinflammatory cytokine IL-10 in monocytes is a major mechanism in the antiinflammatory actions of α-MSH. In the present study, HKC were investigated for their ability to produce IL-10 after α-MSH stimulation. HKC were obtained from the skin of human female breast sections and either left untreated or treated with 0.01 or 0.1 μg/ml α-MSH for different times. Using RT-PCR, HKC were shown to express IL-10 mRNA even under basal conditions, and treatment with α-MSH increased expression. Only minimal concentrations of IL-10 protein were detected in supernatants from the α-MSH-stimulated cultures. To the best of our knowledge this is the first report of IL-10 expression by cultured HKC after α-MSH stimulation. Further studies are needed to determine the biological and therapeutic relevance of these findings. Received: 11 November 1997     

Is there any relationship between serum and urine neopterin and serum interferon‐γ levels in the activity of Behcet's disease?

Background  Behcet's disease (BD) is a chronic, inflammatory, multisystem vasculitic disorder. There is no reliable laboratory marker that indicates disease activity. Neopterin is an immunological marker of cellular immune activation, which is secreted by monocytes/macrophages as a result of interferon-gamma (IFN-γ) secretion by activated T lymphocytes.
Objective  We aimed to investigate serum and urine neopterin levels in BD patients.
Methods  Forty-five patients who were diagnosed according to the criteria of the International Study Group for BD and 45 age- and sex-matched healthy controls were enrolled in the study. Disease activity was considered by clinical findings. Serum and urine neopterin levels and serum IFN-γ levels were measured.
Results  The mean values of serum and urine neopterin levels were 12.68 ± 4.87 nmol/L and 167.53 ± 148.73 µmol/mol creatinine, respectively, in BD patients ( P =  0.000 and P  = 0.008, respectively), which were statistically significantly different from the control group. However, there was no significant statistical difference between serum and urine neopterin levels of the clinically active and inactive patients. It was also found that the mean value of serum IFN-γ levels was higher in healthy controls than in BD patients ( P =  0.000).
Conclusions  We conclude that serum and urinary neopterin measurement can not be used as a reliable laboratory marker as the BD patients' serum and urinary neopterin levels do not increase in the active stage even though these levels increase when compared to healthy controls.     

Dendritic epidermal T cells: activation requirements and phenotypic characterization of proliferating cells

Dendritic epidermal T cells (DETC) are CD45+, Thy-1+, CD5-, CD8-, CD4- murine lymphocytes that express surface-bound CD3 antigens associated with T cell receptor gamma/delta heterodimers. Using epidermal cells greatly enriched for DETC and depleted of Langerhans cells, we found that DETC have growth requirements quite different from those of accessory cell-depleted lymph node and splenic T cells. Although the latter cells strongly proliferate in response to phorbol myristate acetate (PMA) + ionomycin, DETC, when exposed to interleukin-1 (IL-1), interleukin-3 (IL-3), concanavalin A (ConA), PMA, and ionomycin used either alone or in combination, do not exhibit significant mitotic activity. Recombinant interleukin 2 (rIL-2), albeit ineffective by itself, leads to vigorous proliferation of DETC when used with either ConA or PMA + ionomycin + IL-1. In contrast, the combination of PMA and recombinant interleukin-4 (rIL-4), which triggers growth of lymph node T cells, does not induce proliferation of DETC. Although a portion of proliferating DETC expressed CD8 antigens, essentially none bore detectable amounts of surface-bound CD4 or CD5 antigens, or both. Continuing stimulation of primary DETC cultures with lectin/lymphokine-rich media results in the propagation of cells with the essential phenotypic features of resident DETC.     

Ethanol enhances the IFN-γ, TGF-α and IL-6 secretion in psoriatic co-cultures

Summary Evidence suggests an association between alcohol consumption and psoriasis. This relationship is still undefined, although long-term alcohol intake influences the immune system. Interactions between T cells and keratinocytes are important for the pathogenesis of psoriasis, by secretion of proinflammatory cytokines and growth factors in psoriatic skin. IL-2, IL-6, IL-8, IFN-γ and TGF-α are hallmark cytokines in a psoriatic cytokine network. We investigated whether ethanol influences the secretion of these cytokines using a co-culture model with keratinocytes from psoriatic patients (n = 9) or from healthy controls (n = 9), with HUT 78 lymphocytes, and determined the cytokine levels with or without ethanol treatment in the culture supernatants.
TGF-α and IFN-γ levels were elevated in the ethanol-treated psoriatic co-cultures, to 150% and 175% respectively, but neither in co-cultures with keratinocytes derived from healthy control individuals nor in monocultures. Treatment with ethanol elevated slightly the IL-6 levels in the monocultures from psoriatic and control keratinocytes to 125% but not in HUT 78 monocultures. In the psoriatic co-cultures, IL-6 levels were elevated in the culture supernatants to almost 160%, but they were not influenced by ethanol in co-cultures with control keratinocytes. The cytokine levels of IL-8 or IL-2 were not significantly influenced in the psoriatic mono- and co-cultures or in HUT 78 cultures.
If ethanol influences the cytokine secretion of psoriatic keratinocytes and HUT 78 lymphocytes in co-culture conditions, these data suggest that ethanol could also influence the psoriatic cytokine network in vivo, which may explain the aggravation of this disease in alcohol-consuming psoriatic patients.     

Perilesional treatment of metastatic melanoma with interferon-β

Previous trials with various treatments have not shown satisfactory therapeutic results for cutaneous metastasis of malignant melanoma (MM). We report three patients who were treated with peritumoral injection of interferon (IFN)-β for multiple skin metastases of MM. The metastatic tumours were infiltrated by significant numbers of CD8+ TIA+ cytotoxic lymphocytes, and the numbers of CD4+ cells and human leucocyte antigen-DR+ cells increased after IFN-β injection. These results suggest that the peritumoral administration of IFN-β enhanced the antitumour immune response against the MM, suggesting that it is a promising supportive treatment for skin metastasis of MM.     

Regional ganglionar metastasis 24 years after surgical resection of a malignant melanoma

A 40-year-old woman, with type I skin, came to the Dermatology Clinic after noticing a tumor in the armpit. The physical examination revealed a hard, stretched-shaped tumoral node with a diameter of 30 mm, located in the left armpit. It moved on the superficial and deeper layers. In addition, the scar of a large graft was found in the homolateral supraclavicular region, where a malignant melanoma had been excised 24 years earlier. At that time, the primary tumoral lesion had the characteristics of an extensive superficial melanoma with an infiltrating core with vertical growth. The pathologic study of the tumor confirmed the diagnosis of a malignant melanoma with a Clark level II and a 1.17 mm Breslow thickness. Surgery had a margin of 3 cm. At present, the patient is in excellent condition and laboratory tests are normal. Immunologic tests performed showed a slight increase in CD4+, CD45A+, DR+ lymphocytes, CD16+, CD56+, NK cells, and a marked increase in tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) levels (Table 1). Imaging studies (chest X-rays, abdominal ultrasound, bone scintigraphy) did not show any abnormal signs. Computerized tomography confirmed the ganglionic mass, without any other abnormalities. Tc-99m methoxy-isobutyl-isonitrile (MIBI) scintigraphy was performed, showing a focally increased concentration of radiomarker in the left armpit, indicating a thick adenopathic conglomerate (Fig. 1). No other areas of abnormal uptake were observed, not even in the original area of the primary tumor. The surgical exploration of the axillar contents confirmed the presence of adenopathies. Pathology studies showed metastatic invasion of intensely melanin-pigmented and pleomorphic cells, with atypical mitosis.     

Epidermal CD8+ T cells in chronic plaque psoriasis are Tc1 cells producing heterogeneous levels of interferon-gamma

The majority of epidermal CD8+ T cells in chronic plaque psoriasis are activated Tc1 cells producing interferon-gamma and no interleukin-4, a small proportion of which express NK-T receptors. To quantitate their level of cytokine production and characterize them further, CD8+ T cells were isolated from epidermal cell suspensions of lesional biopsies from 24 patients with chronic plaque psoriasis. T-cell lines (TCL) were established by culture of CD8+ T cells with feeders and IL-2 for 11 days and expansion with PHA. Ten TCL were stained for surface markers; 6 were cloned with PHA by limiting dilution. Interferon-gamma, interleukin-4 and interleukin-10 production was measured by ELISA after PMA/anti-CD3 activation of 15 TCL and 39 CD8+ T-cell clones. The 10 TCL stained were CD8alphabeta+ (93.3%), T-cell receptor-alphabeta+ (99.5%), costimulatory molecule CD28+ (90.1%), with a small CD8alphaalpha+ population (2.3%). No NK-T-cell receptor CD158a or CD158b expression was detected, whilst CD94 was expressed on 6.2% of cells in 6/9 TCL. All the TCL and 37/39 CD8+ T-cell clones produced interferon-gamma but no or minimal interleukin-4 or interleukin-10. The TCL produced a wide range of interferon-gamma levels (138 to 15,020 pg/ml). Clones from 3 patients showed low levels (60 to 1,410 pg/ml), from 2 patients high levels (6,105 to 43,040 pg/ml) and from 1 patient a wide range (405 to 36,010 pg/ml) of interferon-gamma production. Thus epidermal CD8+ Tc1 cells in chronic plaque psoriasis produce highly heterogeneous levels of interferon-gamma, which may reflect clinical diversity.     

Glutathione efflux associated with a low γ-glutamyl transpeptidase activity in human melanoma cells

Summary The cellular concentration of reduced glutathione (GSH) modulates the sensitivity of human melanoma cells to alkylating drugs in vitro . To investigate whether the membrane-associated enzyme γ-glutamyl transpeptidase (γ-GTP) involved in GSH breakdown was expressed in melanoma cells. the enzymatic activity of γ-GTP as well as the secretion of GSH were measured in human melanoma cells from four different cell lines (Me8. JUSO, GLL19. Swift). All the cells showed low γ GTP activities (0–1 mU/mg protein) and released GSH in culture supernatants at significant rates. After incubation for 24 h in growth medium containing 0.1 mmol/L cystine, the levels of GSH in supernatants ranged from 56 to 111 nmol GSH/mg protein. The GSH metabolism of melanoma cells was also evaluated by measuring the levels of the melanogenesis intermediate 5-S-cysteinyldopa under different experimental conditions. The results of these experiments suggest that melanoma cells have a low ability to metabolize the tripeptide GSH. which appears to be responsible for GSH secretion and accumulation in culture supernatants.     

In vivo cytokine profiles in allergic and irritant contact dermatitis

Local cytokine profiles in skin biopsies from allergic and irritant patch test reactions were determined by in vivo immunohistochemistry to differentiate between these 2 clinically identical afflictions especially at the time of final reading in diagnostic patch testing. Biopsies were taken from established allergic persons after specific allergic patch test.-, to epoxy resin (1%) and formaldehyde (1%) and from non-allergic individuals with irritant patch tests to sodium lauryl sulfate (10%) and formaldehyde (8%). At 72 h after application of the agents, significantly enhanced frequencies of dermal infiltrating cells, producing IL-1α, TNF-α. IL-2. and IFN-γ per 100 infiltrating cells in the dermis. were observed in allergic as well us irritant patch test reactions, as compared to normal skin. Significantly higher frequencies of IL- Iα-producing cells were observed in biopsies from epoxy resin (1%) allergen-affected and sodium lauryl sulfate (10%) irritant-affected skin as compared to formaldehyde (1%) allergen-affected skin. In addition, significantly higher frequencies of TNF -α reproducing cells were observed in epoxy resin allergen-affected skin us compared to Formaldehyde (1%) allergen-affected and formaldehyde (8%) irritant affected skin. The allergic and irritant patch test reactions showed similar levels of expression of the Thl cytokines IL-2 and IFN-γ in the dermis. confirmed by probe based detection of IL-2 mRNA and IFN-γ- mRNA, In conclusion, the described similarity shows that allergens and irritants can induce the same profile of IL-la. TNF-α. IL-2. and IFN-γ production, resulting in the near impossibility of discriminating between allergic and irritant contact dermal is at the lime of patch test reading.     

高迁移率族蛋白B1对人T淋巴细胞功能性极化的调节作用

目的观察高迁移率族蛋白B1（HMGB1）对人外周血T淋巴细胞功能性极化的影响,并对其作用机制进行初步探讨。方法分离健康人外周血单个核细胞后接种于24孔培养板（2×10^6／ml）,以植物血凝素激活T淋巴细胞体外增殖后,给予不同剂量重组人HMGB1（0、1、10、100和1000ng／m1）进行刺激12、24或48h。采用四色流式细胞术分析CD3^＋淋巴细胞CD4表达和胞内细胞因子干扰素（IFN）-γ、白细胞介素4-（IL-4）阳性（Th1、Th2）的比例。结果不同HMGB1刺激时间和作用剂量对CD4^＋T淋巴细胞和Th1亚群比例未造成明显改变,但1000ng／ml HMGB1刺激48h后CD4^＋／CD3^＋比值显著低于刺激12h组（P〈0．05）。较大剂量HMGB1（100、1000ng／m1）能显著增加Th2亚群比例（P〈0．05或P〈0．01）,并因此降低Th1／Th2比值,出现Th1优势向Th2优势偏移。结论HMGB1剂量蓄积和持续刺激可诱导T细胞功能亚群从促炎反应向抗炎优势转化。HMGB1的这一免疫调控效应可能是机体免疫应答陷人抑制状态,并最终出现免疫麻痹的重要原因之一。     

干扰素γ诱导人黑素细胞CD40的表达

目的 探讨γ-干扰素(IFN-γ)体外诱导人黑素细胞表面CD40分子的表达及其意义.方法 常规分离培养人黑素细胞,流式细胞仪测定IFN-γ处理前后细胞表面CD40等免疫分子表达;混合淋巴细胞反应评价黑素细胞对同种异体T淋巴细胞的刺激能力;ELISA检测培养上清液IL-8、IL-10、IL-12的浓度.结果 体外培养的人黑素细胞表面表达少量CD40分子;不同浓度的IFN-γ处理黑素细胞24h、48h、72h后,能显着促进其CD40的上调(P<0.01),且24h处理组的黑素细胞CD40的表达量和IFN-γ的浓度呈直线相关.经IFN-γ诱导后的黑素细胞形态有所变化,刺激同种异体淋巴细胞的能力也显着增加,300IU/mLIFN-γ处理的黑素细胞72h刺激指数(SI)可达到峰值.黑素细胞经IFN-γ作用后,培养上清液中IL-12水平明显增加(P<0.05),而IL-8、IL-10的浓度无变化(P>0.05).经IFN-γ预处理的黑素细胞经SCD40L配基化后,能显着上调CD80、细胞间粘附分子1(P<0.01),且这种作用能被特异性的CD40L的单克隆抗体所阻断.结论 IFN-γ体外能够诱导黑素细胞功能性地表达CD40分子及增加对淋巴细胞的刺激能力,这对于认识黑素细胞在细胞免疫应答中的作用具有重要的意义,CD40分子上调后,黑素细胞可能不经过CD4⁺细胞而直接刺激活化CD8⁺的杀伤性T细胞(CTL).     

流式细胞仪测量组胺对人角质形成细胞内游离Ca~(2+)的影响

目的探讨组胺对人角质形成细胞(HKC)胞内游离Ca2+浓度的影响。方法利用流式细胞仪(FCM)结合Fluo3染色技术,半定量检测组胺及H2组胺受体拮抗剂西咪替丁对体外培养HKC胞内Ca2+浓度的影响。结果组胺以剂量依赖方式引起HKC胞内Ca2+浓度上升,西咪替丁则完全阻断组胺该作用。结论组胺通过H2组胺受体激活HKC相关信号传导系统,从而引起细胞内Ca2+浓度增加。