Regulation of the expression of aminopeptidase A,aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators

Aminopeptidase (AP) A is a transmembrane type II molecule widely distributed in mammalian tissues. Since APA expression may be absent in renal cell carcinoma (RCC), it is possible that there is an altered regulation or other defect of APA upon malignant transformation of proximal tubular cells. However, investigations into the regulation of APA on tumour cells are rare. We report, for the first time, that both transforming growth factor-beta 1 (TGF-β1) and tumour necrosis factor-alpha (TNF-α) down-regulate APA mRNA as well as protein expression in renal tubular epithelial cells and RCC cells in culture. In addition to this, both cytokines decrease dipeptidylpeptidase (DP) IV/CD26 mRNA, but not APN/CD13 mRNA expression. Otherwise, IL-4 and IL-13 increase CD13 as well as CD26 expression, but do not alter APA expression. Interferon-alpha (IFN-α), IFN-β and IFN-γ increase mRNA expression of all the three membrane ectopeptidases, whereas IL-1, IL-6, IL-7, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any significant effect. Treatment of cultured cells with cAMP-increasing agents, such as 8-bromo-cAMP or A23187, results in an increase in APA and DPIV/CD26, but no change in APN/CD13 mRNA expression or even a decrease in it. Furthermore, AP inhibitors can influence APA mRNA expression, since bestatin causes an increase in APA expression in a time- and dose-dependent manner, whereas bestatin does not change CD13 or CD26 expression. No difference could be found with respect to the modulation by different mediators between RCC cells and renal epithelial cells, though permanent tumour cell lines such as Caki-1 and Caki-2 may have lost some of the normally expressed peptidases.     

Enrichment of human beta 1 bri/alpha 6 bri/CD71 dim keratinocytes after culture in defined media

Stem cell-like keratinocytes are responsible for the high regenerative potential of the skin. For clinical applications using keratinocytes in artificial skin constructs, it is suitable to work with serum-free medium under defined conditions. This is also true for the preceding expansion of the stem cell-like keratinocyte population. Therefore, we analyzed the effect of a serum-free medium on the population distribution in comparison to an established serum-containing standard medium for keratinocyte culture. We quantified the freshly isolated as well as cultured primary human keratinocytes by their expression of the beta(1) integrin (CD29) in combination with the expression of the alpha(6) integrin (CD49f) and the transferrin receptor (CD71) by flow cytometric methods. We were able to show that cultivation with serum-free medium induces a switch of the cell population to higher expression of the beta(1) integrin. In addition, the proportion of the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population was enhanced about 35.4 +/- 6.56% after cultivation with serum-free medium. Culture in serum-containing medium increased this proportion of the keratinocyte cell population only about 17.3 +/- 8.06%, when compared to the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population measured directly after isolation. Our data show that the applied culture conditions already have an enormous impact on the development of a stem cell-like phenotype of keratinocytes. This work demonstrates that the serum-free medium significantly increases the proportion of beta(1)(bri)/alpha(6)(bri)/CD71(dim)-expressing keratinocytes. In conclusion, these findings implicate new applications in keratinocyte stem cell research and regenerative medicine.     

Increased expression of neutral endopeptidase (NEP) and aminopeptidase N (APN) on peripheral blood mononuclear cells in patients with multiple sclerosis

We studied CD10 Ag of neutral endopeptidase (NEP) and CD13 Ag of aminopeptidase N (APN) expression on peripheral blood mononuclear cells (PBMC) as cells activation markers and for their transendothelial migration properties in three groups of MS patients (total 58); with acute exacerbation of MS (n = 18), primary progressive MS (n = 17) and with MS remission (n = 23). The control group (OND) consisted of 24 patients, suffering from other noninflammatory neurological diseases. CD10 Ag and CD13 Ag expression on PBMC was higher in clinically active MS (acute exacerbation and progressive MS) compared to MS remission and OND groups. Our study suggests that CD10 Ag and CD13 Ag can be useful mononuclear cell activation markers in the course of MS. CD13 Ag expression on PBMC may be also the sensitive marker of these cells transendothelial migration properties.     

IL-10 and TGF-beta differ in their regulation of aminopeptidase N/CD13 expression in monocytes

Human monocytic cells express considerable amounts of aminopeptidase N (APN)/CD13, a transmembrane protein proposed to play a role in the modulation of kinins, neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Previous studies have shown that APN/CD13 participates in antigen processing and presentation, trimming peptides protruding out of MHC class II molecules. In several inflammatory processes, macrophages have been shown to express especially high amounts of MHC class II molecules and of this peptidase. To learn more about the regulation of APN/CD13 on monocytes we investigated its expression under the influence of cytokines. Here, we report a dose- and time-dependent up-regulation of APN/CD13 mRNA and protein expression by transforming growth factor (TGF)-beta on human monocytes. To the contrary, we found IL-10 down-regulating the expression of APN/CD13 mRNA and protein. Both the regulation of the APN/CD13 protein assessed by immunofluorescence and the gene expression assessed by real-time PCR were highly correlated. Using the Dual-Luciferase reporter assay, we demonstrate that TGF-beta treatment of monocytes results in a higher activity of the APN/CD13 myeloid promoter. Our results implicate differences in the expression of the membrane peptidase APN/CD13 and therefore in the peptide-modulating ability of monocytes after exposure to these two immunosuppressive cytokines, TGF-beta and IL-10.     

Elevated synovial tissue concentration of the common acute lymphoblastic leukaemia antigen (CALLA)-associated neutral endopeptidase (3.4.24.11) in human chronic arthritis.

The cell-surface neutral endopeptidase 3.4.24.11 (NEP) activity of the common acute lymphoblastic leukaemia antigen (CALLA) cleaves diverse peptide mediators at specific sites and it has been postulated that it regulates immune responses. The concentration of NEP was quantified in detergent extracts of synovial tissues by the percentage hydrolysis of [3H-D-Ala]-Leu enkephalin/hr/100 mg of tissue. The synovial tissue concentration of NEP was higher in all patients with rheumatoid arthritis (n = 7; group mean +/- SD = 29.4 +/- 20.2%), and was higher with degenerative joint disease (n = 6 of 8; group mean +/- SD = 11.9 +/- 10.4%) than with traumatic arthropathy (n = 3; 1.1 +/- 0.7%). The lack of direct relationship between synovial tissue NEP concentration and leukocytic infiltration suggests that the cellular source of NEP may be synoviocytes or fibroblasts, and that NEP may have distinctive pathogenetic roles in human arthritis.     

Toxic epidermal necrolysis and Stevens-Johnson syndrome are induced by soluble Fas ligand

The pathogeneses of toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS), both severe blistering diseases usually associated with drug intake, are not fully elucidated. Histologically, both TEN and SJS are characterized by extensive keratinocyte apoptosis. Previous studies have shown that keratinocyte apoptosis in TEN and SJS was induced by a suicidal interaction between Fas and Fas ligand (FasL), which are both expressed by keratinocytes. However, our preliminary examinations demonstrated that FasL is hardly detected on keratinocytes. We hypothesized that soluble FasL (sFasL) is secreted by peripheral blood mononuclear cells (PBMCs), and this interacts with the Fas expressed on keratinocytes in TEN and SJS. To justify this hypothesis, we investigated whether sFasL secreted by PBMCs could induce the keratinocyte apoptosis in TEN and SJS. Enzyme-linked immunosorbent assay analysis demonstrated that there was no significant sFasL increase in any samples of healthy controls (<40 pg/ml, n = 14) and patients with an ordinary erythema multiforme-type drug eruption (41.5 +/- 3.1 pg/ml, n = 14), whereas high concentrations are detected in all samples of TEN and SJS patients (TEN: 131.5 +/- 57.4 pg/ml, n = 8; SJS: 119.1 +/- 41.0 pg/ml, n = 14) (P < 0.0001). In vitro analysis using cultured keratinocytes revealed that the sera of TEN and SJS patients induced abundant keratinocyte apoptosis compared to erythema multiforme-type drug eruption sera. Furthermore, on stimulation with the causal drug, PBMCs obtained from TEN and SJS patients secreted high levels of sFasL. Taken together, these results indicate that sFasL secreted by PBMCs, not keratinocytes, plays a crucial role in the apoptosis and pathomechanism of TEN and SJS, and that the serum sFasL level may be a good indicator for the early diagnosis of TEN and SJS.     

乳鼠角朊细胞分离表达共刺激分子CD80/CD86

以往研究认为角朊细胞不表达共刺激分子CD80／CD86，无法提供淋巴细胞增殖过程中关键的共刺激信号，但培养的角朊细胞膜片移植后，最终供者细胞被完全排斥，具体机理不明。本实验使用近交系小鼠，有和无血清两种体系培养角朊细胞1、4、7d，采用流式细胞技术、激光共聚焦技术，RT-PCR方法检测角朊细胞在培养过程中MHCⅠ类、Ⅱ类抗原，及CD80、CD86的表达。证实培养的细胞中不含郎格罕氏细胞，首次发现乳鼠角朊细胞在培养过程中表达CD80，但不表达CD86，可刺激异基因淋巴细胞增殖，执行抗原递呈细胞功能。本研究结果为角朊细胞膜片移植排斥提出另一种可能的解释：MHCⅠ／Ⅱ类分子和CD80的表达，使培养的异体角朊细胞兼具外来抗原和抗原递呈细胞的功能，刺激受体淋巴细胞增殖，参与排斥反应。     

In vivo optical imaging of CD13/APN-expression in tumor xenografts

The metalloexopeptidase CD13/aminopeptidase N (APN) has been shown to be involved in cancer angiogenesis, invasion, and metastasis. Therefore, a CD13/APN-targeted NGR-peptide was labeled with the cyanine dye Cy 5.5 and applied to image tumor xenografts with different APN-expression levels using both planar and tomographic optical imaging methods. In vitro, the peptide-dye conjugate showed a clear binding affinity to APN-positive HT-1080 cells, while negative MCF-7 cells and predosing with the free NGR-peptide revealed little to no fluorescence. In vivo, tumor xenografts (n>or=5) were clearly visualized by two-dimensional (2-D) planar fluorescence reflectance imaging (FRI) and three-dimensional (3-D) fluorescence mediated tomography (FMT) up to 24 h after injection. FMT also allowed us to quantify fluorochrome distribution in deeper tissue sections, showing an average fluorochrome concentration of 306.7+/-54.3 nM Cy 5.5 (HT-1080) and 116.0+/-18.3 nM Cy 5.5 (MCF-7) in the target tissue after 5 h. Competition with the free NGR-peptide resulted in a reduction of fluorochrome concentration in HT-1080 tumor tissue (195.3+/-21.9 nM; 5 h). We thus conclude that NGR-Cy 5.5 combined with novel tomographic optical imaging methods allows us to image and quantify tumor-associated CD13/APN expression noninvasively. This may be a promising strategy for a sensitive evaluation of tumor angiogenesis in vivo.     

Assessment of an automated bioreactor to propagate and harvest keratinocytes for fabrication of engineered skin substitutes

Engineered skin substitutes (ESS) composed of autologous fibroblasts and keratinocytes attached to collagen-glycosaminoglycan (GAG) scaffolds are effective adjuncts in the treatment of massive burns. The Kerator, an automated bioreactor for keratinocyte culture, could hypothetically reduce labor and material requirements, and increase availability of ESS. Human keratinocytes were cultured in the Kerator and also in tissue-culture flasks. It was found that keratinocyte confluence increased exponentially with time in both the Kerator (r2=0.99) and the flasks (r2=0.96). Confluence (mean+/-SEM) of keratinocytes in the flasks (28+/-2.3%) was significantly higher than in the Kerator (18+/-0.93%) at day 4. However, there was no difference in confluence at harvest. The colony forming efficiency (CFE) and population doublings (PD) per day of keratinocytes harvested from the Kerator were 67+/-4.7% and 0.80+/-0.06, respectively, and were not different from the corresponding values for keratinocytes from flasks. ESS fabricated with keratinocytes from the Kerator or from the flasks were comparable in vitro in terms of histological anatomy, cellular viability, and surface hydration. These findings show that there are no differences between keratinocytes from the Kerator and those from the flasks regarding (a) growth to confluence, (b) CFE and growth rate (PD/day), or (c) quality of ESS in vitro, suggesting that the Kerator can automate fabrication of ESS and increase its availability for treatment of skin wounds.     

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.     

Effect of neutral endopeptidase inhibition on substance-P-induced increase in short-circuit current of canine cultured tracheal epithelium.

We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussing's short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP.     

Cultured keratinocyte grafts are recognized, but not rejected by CD8+ T cells in vivo

It has been shown in the mouse that cultured keratinocyte allografts [fully major histocompatibility complex (MHC) mismatched] survive for at least 100 days without evidence of rejection. In an attempt to analyze the immune mechanisms underlying this phenomenon we have investigated the induction of tolerance to such grafts. Primary cultures of BALB/c keratinocytes were prepared using irradiated 3T3 feeder cells, and the cultured cell sheets were grafted, using a silicone transplantation chamber, onto CBA recipients. After the cultured grafts had been in place for 4-6 weeks full-thickness tail skin from BALB/c donors was grafted onto the dorsal flank opposite the cultured graft. The median graft survival time of these full-thickness allografts was 15.5 days compared to 13 days in the control group. These data show that in the absence of Langerhans cells and MHC class II expression, keratinocytes expressing class I and minor histocompatibility antigens induce a prolonged survival of full-thickness skin allografts. The results from experiments in which T cell subsets were depleted in vivo suggest that CD8+ cells (a) recognize the class I alloantigens of the cultured graft, (b) do not reject the cultured graft and (c) do not progress further in their maturation pathway. We propose that these CD8+ T cells might have been partially primed by receiving only the first signal of activation and that they may be equivalent to "poised" cytotoxic T cells. These CD8+ cells can reject non cultured full-thickness skin grafts, and do so more effectively after removing CD4+ cells.     

High levels of functional endopeptidase 24.11 (CD10) activity on human thymocytes: preferential expression on immature subsets.

Although it is now well established that cells of the immune system express most of the exopeptidases described so far, little information is available concerning the identification and the characterization of the peptidases associated with the surface of human thymocytes. In the present study we have focused on CD10 expression on thymocytes using both FACS and enzymatic analysis. Unfractionated intact human thymocytes were shown to express significant levels of CD10-specific enzymatic activity, as assessed by the hydrolysis of the neutral endopeptidase (NEP) substrate Suc-Ala-Ala-Phe-pNA and of D-Ala2-Leu-enkephalin, a typical NEP substrate. CD10 activity was abolished by specific NEP inhibitors, including thiorphan, retrothiorphan and phosphoramidon. Moreover, high performance liquid chromatography (HPLC) analysis showed that intact thymocytes and purified NEP hydrolysed thymopentin, a thymic factor known to induce the maturation of prothymocytes into thymocytes. Finally, CD 10/NEP was preferentially associated with CD3- CD3low and immature CD4- CD8- thymocytes. The data demonstrate for the first time that human thymocytes express functional NEP and suggest a role for this enzyme in the maturation of human thymocytes.     

Integrin CD11c contributes to monocyte adhesion with CD11b in a differential manner and requires Src family kinase activity

Integrin-mediated adhesion of human monocytes to fibrinogen regulated by CD11b/CD18 and the closely related integrin CD11c/CD18, play a key role in inflammation. Peripheral blood monocytes isolated from human donors despite expressing CD11c primarily utilized CD11b to mediate adhesion to fibrinogen upon stimulation with granulocyte macrophage-colony stimulating factor (GM-CSF) and fMLP. Blocking with anti-CD11b resulted in 90% (p<0.001, n=3) inhibition of monocyte adhesion. Monocytes cultured in human serum showed a shift in the participation of integrins, adhesion to fibrinogen involving both CD11b and CD11c. The participation of CD11c in cultured monocytes corresponded to a 3.4-fold increase in expression in CD11c. Blocking cultured monocytes with anti-CD11b or anti-CD11c alone showed no significant effect on adhesion. Treatment with both anti-CD11b and anti-CD11c resulted in inhibition of adhesion by 85% (p<0.001, n=3). Abrogation in adhesion upon treatment with PP1 or PP2 showed that Src family kinase activity was required for CD11b and CD11c mediated adhesion of cultured monocytes to fibrinogen upon stimulation with GM-CSF and fMLP. The clustering of CD11c on cultured monocytes upon adhesion to fibrinogen was diminished on inhibition with PP2 indicating a role for Src family kinase activity in regulating CD11c avidity. CD11b was critical to cytoskeletal events leading to increased spreading and formation of actin foci in cultured monocytes following adhesion to fibrinogen. Blocking cultured monocytes with anti-CD11b or anti-CD11c alone showed that the increase in spread area was diminished by 67+/-3% and 36+/-9%, respectively. The differential involvement of CD11c and CD11b in adhesion and subsequent cytoskeletal changes in monocytes exposed to different conditions indicates the importance of each integrin in distinct responses during inflammation.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Substance P induced preprotachykinin-a mRNA,neutral endopeptidase mRNA and substance P in cultured normal fibroblasts

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1-3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30-40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases.     

The antigen-presenting environment in normal and human papillomavirus (HPV)-related premalignant cervical epithelium

The activation of HPV-specific T cells within the cervical microenvironment is likely to play an important part in the natural history of cervical intraepithelial neoplasia (CIN). The extent and the type of T cell activation will depend critically on the expression of MHC, costimulatory cell surface molecules and cytokines by keratinocytes and Langerhans cells within the cervical lesion. Expression of MHC class II (HLA-A-DR and -DQ), costimulatory/adhesion molecules (CD11a/18, CD50, CD54, CD58 and CD86) and cytokines (tumour necrosis factor-alpha (TNF-alpha) and IL-10) was therefore investigated by immunohistochemistry in normal squamous epithelium (n = 12), low-grade (n = 23) and high-grade (n = 18) squamous intraepithelial lesions of the cervix. CIN progression was associated with de novo expression of HLA-DR and CD54, and increased expression of CD58 by keratinocytes. However, significantly, there was no expression of any adhesion/costimulation molecule by epithelial Langerhans cells in any cervical biopsy studied. Furthermore, TNF-alpha, a potent activator of Langerhans cells, was expressed constitutively by basal keratinocytes in normal cervix (12+/12). but expression of this cytokine was absent in a number of CIN samples (20+/23 for low-grade, 12+/18 for high-grade CIN). Conversely, the suppressive cytokine IL-10 was absent in normal epithelium (0+/12), but was up-regulated in a number of CIN lesions (12+/23 for low-grade; 8+/18 for high-grade CIN). The restricted expression of costimulation/adhesion molecules and the nature of the cytokine microenvironment within the epithelium may act to limit effective immune responses in some CIN lesions.