Immunohistochemical study of inducible nitric oxide synthase in skin cancers

BACKGROUND: Nitric oxide (NO) is synthesized from the amino acid L-arginine by NO synthase (NOS). Experimental evidence suggests that increased express of inducible NOS (iNOS), which is an NOS isoform and calcium independent, is related to various pathological processes, such as inflammation and cancer. METHODS: In this study, we used immunohistochemistry to investigate iNOS expression in a series of basal cell carcinomas (BCC), Bowen's disease, squamous cell carcinomas (SCC), extramammary Paget's disease (EPD) and metastatic tumors of the skin. RESULTS: Only 1 of 16 BCC cases was positive for iNOS and the intensity of staining was weak. In most of the 10 cases of Bowen's disease, iNOS was weakly expressed and there was a wide range in the percentage of positive tumor cells. Twelve of the 16 cases of SCC were positive for iNOS and the extent of positivity was greater than in Bowen's disease. Two of the 7 cases of EPD were positive for iNOS, and 12 of the 15 cases of metastatic cancer were positive. Well-differentiated adenocarcinomas were diffusely positive, whereas poorly-differentiated ones showed strong and heterogeneous staining. CONCLUSIONS: These results indicated that the expression of iNOS may reflect the proliferation of tumor cells and that a heterogeneous distribution of iNOS may correlate with a wide variety of biological behavior of tumor cells.     

Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin

Kang-Rotondo CH, Major S, Chiang TM, Myers LK, Kang ES. Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin. Photodermatol Photoimmunol Photomed 1996: 12: 57–65. © Munksgaard, 1996. Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [³H]l -citrulline using labeled l -arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2–48.3 mJ/cm² but was inhibitory after 64.4 and 80.5 mJ/cm². Thirty min after 10^?6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M_r 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.     

皮肤恶性黑素瘤中诱导型一氧化氮合酶mRNA及其蛋白的表达

目的探讨皮肤恶性黑素瘤中诱导型一氧化氮合酶（iNOS）的表达及其临床意义。方法免疫组化方法检测30例皮肤恶性黑素瘤患者肿瘤组织中iNOS蛋白的表达，逆转录聚合酶链反应（RT-PCR）检测其中6例患者肿瘤组织和肿瘤邻近正常组织中iNOSmRNA的表达。结果iNOS在肿瘤组织中无论蛋白或mRNA阳性表达率皆明显高于邻近正常组织（P〈0．05）。其中iNOS蛋白在无转移的恶性黑素瘤中阳性表达率为69．2％，有转移的恶性黑素瘤中阳性表达率为100％。结论iNOS表达异常在恶性黑素瘤发生、发展与转移过程中可能起重要作用。     

The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes

Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.     

Constitutive endothelial and inducible nitric oxide synthase in inflammatory dermatoses

We have used immunohistochemistry to localize the expression of the constitutive endothelial and inducible forms of the enzyme nitric oxide synthase (NOS) in skin from involved and uninvolved sites in patients with atopic dermatitis (AD) and allergic contact dermatitis (CD). Endothelial NOS (eNOS) immunoreactivity was localized to vascular endothelium in the dermis of both involved and uninvolved skin from all patients. Inducible NOS (iNOS) immunoreactivity was found to be closely associated with the upper dermal microvasculature in all the involved AD biopsies, but only in two of 10 uninvolved AD biopsies. CD biopsies were taken from 10 positive skin patch test sites and iNOS immunoreactivity was detected in all of these. iNOS immunoreactivity was detected in only one of the negative patch test biopsies. Both the extent and intensity of iNOS immunoreactivity was lower in CD than in AD skin lesions. The presence of eNOS in the skin is necessary for constitutive NO-mediated dilatation of the dermal vasculature. Induction of iNOS in the dermal endothelium and in perivascular inflammatory cells may be significant with respect to the roles of NO in both the vasodilatory component of the inflammatory response and in the modulation of immune responses in the skin.     

UVB radiation-mediated expression of inducible nitric oxide synthase activity and the augmenting role of co-induced TNF-alpha in human skin endothelial cells

Nitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-alpha expression. In HUDEC addition of TNF-alpha results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-alpha- dependent mechanism which essentially contributes to increased iNOS activity.     

Expression of the inducible isoform of nitric oxide synthase in pigment cell lesions of the skin

Nitric oxide (NO) is a small molecule produced during the conversion of L-arginine to L-citrulline by NO synthase (NOS). Several isoforms of NOS exist, of which the Ca2+-independent, inducible NOS (iNOS or NOS2) is most prominently expressed by macrophages. iNOS activity and increased levels of iNOS have been found in various tumours and tumour cell lines but not in normal tissues; however, the precise role of NO in tumour progression has yet to be elucidated. We studied the expression of iNOS in paraffin sections of 41 benign naevi and 52 primary malignant melanomas (MM) of the skin, as well as in 13 metastatic MM. In addition, nitrotyrosine, indicative of NO production and formation of peroxynitrite, was studied in frozen sections of 13 naevi and 30 MM. Virtually all naevi expressed iNOS, but very few expressed nitrotyrosine, indicating either that iNOS in naevi is functionally inactive, or that naevus cells lack reactive oxygen radicals and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In MM, iNOS was most frequently expressed in the 'pure' and 'invasive' radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in 76% of cases; moreover, in these latest phases of tumour progression, iNOS staining was weak and focal. We conclude that iNOS is expressed de novo in most benign pigment cell lesions. In MM (iNOS-generated) NO appears to play an important part in the early steps of invasion (i.e. the 'invasive' RGP), where it may stimulate neo-angiogenesis and may be a prerequisite for further tumour progression; this view is also supported by the finding of iNOS in the associated blood vessels in the papillary dermis. Finally, our data suggest that (iNOS-generated) NO plays a less significant part in the VGP and in metastatic melanoma.     

长波紫外线照射对HaCaT细胞iNOS/NO表达的影响

目的 探讨不同剂量长波紫外线 （UVA） 照射HaCaT细胞后不同时间点诱导型一氧化氮合成酶（iNOS）的表达情况。方法 1 J/cm2、5 J/cm2和10 J/cm2 UVA照射HaCaT细胞后继续培养24 h、48 h和72 h,倒置相差显微镜下观察细胞形态的变化;分别用RT-PCR、Western印迹和Griess法检测HaCaT细胞iNOS mRNA、蛋白及NO的表达。结果 所有UVA剂量组HaCaT细胞iNOSmRNA在光照后24 h有表达,48 h达高峰,72 h后下降,各时间点间表达量差异有统计学意义（P < 0.05）;1 J/cm2 UVA照射后3个时间点均未见iNOS蛋白表达,而5 J/cm2和10 J/cm2 UVA照射后iNOS蛋白在24 h增加,48 h达高峰且显著高于24 h（P < 0.05）,照射后72 h无iNOS蛋白表达。所有UVA剂量组HaCaT细胞NO表达量在24 h升高,48 h显著升高,72 h平稳升高,3个时间点NO表达量均比正常对照组明显增加（P < 0.05）。对照组HaCaT细胞无iNOS mRNA和蛋白表达,NO表达量低。结论 HaCaT细胞iNOS和NO的表达变化与UVA照射存在时间和剂量关系。     

Increased elongated and stabilized microtubules in psoriatic keratinocytes

Summary Microtubules (MT) occur in the keratinocytes as 20 nm broad tubular structures with an electron-lucent core surrounded by an electron-dense wall. The number and the length of the MT increased to the upper epidermal layers.In the normal keratinocytes the MT were relatively short (maximal length 2.2 m) and slightly tortuous. Their outline was tenuous and irregular. Sometimes the MT were interrupted indicating a high fragility. In the upper layers of the psoriatic epidermis the MT reached a maximal length of 3.1 m, showing a greater elongation than in the normal epidermis. The MT were straight and distinctly limited, most likely resulting from a higher grade of stabilization or polymerisation.By their interaction with the tonofilaments the MT may contribute to the stability of the cell shape, delaying the flattening of the psoriatic keratinocytes in the upper epidermal layers. Nowhere did the MT make direct contact with the cell membrane. At some places they did gain access to the cell membrane by short microfilaments.Presented at the 5th European Meeting on Electron Microscopy Applied to Cutaneous Pathology, Copenhagen, May 12–13, 1978     

Expression of inducible nitric oxide synthase and nitrotyrosine in borderline leprosy lesions

BACKGROUND: In the response to T-helper cell (Th1)-type cytokines and interactions with pathogens, high levels of nitric oxide (NO) are produced by activated macrophages expressing the inducible NO synthase (iNOS). The role and importance of reactive nitrogen intermediates (RNIs) such as NO and peroxynitrite in the host response to diseases caused by intracellular pathogens such as Mycobacterium leprae and M. tuberculosis is unclear. OBJECTIVES: The aim of this study was to investigate the presence of local production of NO and peroxynitrite in borderline leprosy by using antibodies against iNOS and the product of peroxynitrite, nitrotyrosine (NT). METHODS: We detected the presence of iNOS and NT in skin biopsies from borderline leprosy patients, with and without reversal reaction (RR), by immunohistochemistry (n = 26). RESULTS: In general, the granulomas from borderline leprosy lesions with and without RR showed high and specific expression of iNOS and NT. Moreover, strong immunoreactivity to iNOS and NT was observed in granulomas surrounding and infiltrating dermal nerves. The expression of iNOS and NT was also strong in keratinocytes, fibroblasts and endothelial cells in close relation to the granulomatous reaction. In contrast, normal human skin showed no expression of iNOS and NT in these cells. CONCLUSIONS: We conclude that iNOS and NT are expressed in granulomas from borderline leprosy patients with and without RR and propose that RNIs might be involved in the nerve damage following RR in leprosy.     

Lipopolysaccharide and cytokines induce nitric oxide synthase and produce nitric oxide in cultured normal human melanocytes

Abstract The role of nitric oxide in normal and pathological conditions of human skin is still poorly understood. In this study we have demonstrated by immunobloting the expression of an inducible nitric oxide synthase isoform (iNOS) in cultured normal human melanocytes treated with bacterial lipopolysaccharide, tumor necrosis factor-· and interferon-á. Nitric oxide was also detected in the culture medium and its formation was abolished upon treatment with N^G-monomethyl-l-arginine(l-NMMA), a competitive inhibitor of nitric oxide synthase. These results suggest that nitric oxide could led to autodestruction of melanocytes causing skin depigmentation. The therapeutic relevance of nitric oxide synthase inhibitors in treatment of vitiligo was suggested. Received: 15 September 2000 / Revised: 7 October 2000 / Accepted: 20 January 2001     

Pemphigus serum and captopril induce heat shock protein 70 and inducible nitric oxide synthase overexpression, triggering apoptosis in human keratinocytes

BACKGROUND: Captopril is an angiotensin-converting enzyme inhibitor with sulphydryl groups in its chemical structure. It is commonly used as an antihypertensive drug. The occurrence of pemphigus vulgaris has repeatedly been reported in patients receiving captopril. The capacity of captopril and pemphigus serum to induce acantholysis, in vivo or in vitro, has been demonstrated experimentally. OBJECTIVES: To show that captopril and pemphigus serum, acting by a biochemical and immunological mechanism, respectively, trigger apoptosis. METHODS: Human keratinocyte cells were treated with 15 mmol L-1 captopril or with pemphigus serum. DNA was extracted and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method was used to detect apoptosis. RESULTS: DNA fragmentation occurred after 72 h of treatment. Increased expression of p53, c-myc and inducible nitric oxide (NO) synthase (iNOS) mRNA were observed by polymerase chain reaction (PCR) in the treated cells compared with the untreated ones. The increase in iNOS gene expression was associated with overproduction of NO. Moreover, the addition of 1 mmol L-1N-monomethyl-L-arginine, a structural analogue of arginine, reduced nitrite levels by about 70% in cells treated with captopril or pemphigus serum. Western blot analysis revealed an overexpression of heat shock protein 70 (hsp70) in cells treated with captopril or pemphigus serum. Finally, total inhibition of the keratinocyte transglutaminase gene was shown by PCR analysis in the same samples, compared with control cells. CONCLUSIONS: These data demonstrate the involvement of apoptosis in keratinocytes treated with captopril or pemphigus serum, with induction of the iNOS gene and hsp70 in the cascade of events leading to programmed cell death.     

Effect of polyamine antimetabolites on cultured human keratinocytes from normal and uninvolved psoriatic skin

We describe the effect of two polyamine antimetabolites on polyamine and macromolecule synthesis of cultured human keratinocytes obtained by suction blisters from normal skin and the uninvolved skin of psoriatic patients. The concentrations of spermidine and spermine steadily increased during the culture of normal keratinocytes in vitro, whereas the putrescine concentration showed only a transient rise at the beginning of the active growth phase. Treatment with difluoromethylornithine decreased the concentrations of putrescine and spermidine in both normal and uninvolved psoriatic keratinocytes, but had no effect on either DNA or protein synthesis. Methylglyoxal bis(guanylhydrazone) marginally decreased the levels of spermidine and spermine and significantly inhibited the DNA and protein synthetic activities. Pretreatment of uninvolved psoriatic keratinocytes with difluoromethylornithine enhanced the accumulation of methylglyoxal bis(guanylhydrozone), resulting in a profound inhibition of cellular macromolecule synthesis. This synergistic was not seen in normal keratinocytes. Thus, although no statistically significant difference was observed between the cells derived from normal and uninvolved psoriatic epidermis, the psoriatic keratinocytes appeared to be more sensitive to the action of polyamine antimetabolites. The inhibition of DNA and protein synthesis by methylglyoxal bis (guanylhydrazone) was prevented by concomitant treatment with spermidine.     

Human dermal microvascular endothelial cells express inducible nitric oxide synthase in vitro

Stimulation of inducible nitric oxide synthase with subsequent release of nitric oxide in large amounts may play a critical part either in host defense reactions or in the pathophysiology of the inflammatory response syndrome leading to septic shock. The aim of the present study was to investigate whether human dermal microvascular endothelial cells exhibit the typical characteristics of an inducible nitric oxide synthase expressing cell. A strong effect on inducible nitric oxide synthase gene expression could be detected when the cells were coincubated with the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha with inducible nitric oxide synthase cDNA concentrations averaging 11.7 +/- 0.6 amol per microg total RNA at 24 h, and 25.0 +/- 1.4 amol per microg total RNA at 48 h, respectively. Intracellular staining with an antibody recognizing inducible nitric oxide synthase protein and subsequent analysis by flow cytometry revealed a 4-fold increase of inducible nitric oxide synthase protein in human dermal microvascular endothelial cells treated with interferon-gamma/tumor necrosis factor-alpha. This was accompanied by a significant elevation in nitrite/nitrate concentrations in the cell-free culture supernatants. Our results indicate that human dermal microvascular endothelial cells are provided with an inducible nitric oxide synthase system and can be regarded as an appropriate cell model for investigating inducible nitric oxide synthase gene expression and nitric oxide properties in microvascular endothelial cells.     

Abnormal maturation pathway of keratinocytes in psoriatic skin

We compared the maturation pathway of normal and psoriatic epidermis using three different markers: (1) Involucrin, which is normally detected in the stratum granulosum in normal skin, was detected in all but the basal layer of involved psoriatic skin; (2) an antigen, recognized by the murine monoclonal antibody psi 3, was present in all but the basal layer of involved psoriatic skin but was absent from uninvolved and normal skin; (3) fibronectin, which normally localizes in the dermis and the epidermal-dermal junction, was also detected intra- and extracellularly in the psoriatic epidermis. These results indicate that the alterations in keratinocyte maturation found in psoriasis do not arise from a truncation of the normal maturation pathway but rather reflect the onset of an abnormal pathway of differentiation characterized by the expression of psi 3 antigen and fibronectin and the premature appearance of involucrin.