U. Junker

Procalcitonin expression in human peripheral blood mononuclear cells and its modulation by lipopolysaccharides and sepsis-related cytokines in vitro.

Procalcitonin (PCT), the precursor of calcitonin, was recently put forward as a diagnostic marker of systemic bacterial infection and sepsis. The major PCT production site in sepsis still remains unclear. Because of a certain association between increased levels of PCT and leukocyte-derived cytokines during sepsis, we assessed the possible expression of PCT in human peripheral blood mononuclear cells (PBMCs) and the modulation of PCT by lipopolysaccharides (LPS) and various sepsis-related cytokines by reverse transcriptase-polymerase chain reaction (RT-PCR) by using a novel primer set and flow cytometric analysis with intracellular staining with antibodies to the PCT components calcitonin and katacalcin. RT-PCR and flow cytometric analysis demonstrated that PBMCs express PCT both on mRNA and on protein levels. LPS and various proinflammatory cytokines (interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha), IL-2) had pronounced stimulatory effects on the expression of PCT mRNA. Under identical experimental conditions the anti-inflammatory cytokine IL-10 had no effect on the expression of mRNA for PCT. Flow cytometric analysis demonstrated increased intracellular amounts of PCT components after LPS stimulation. Thus we demonstrate for the first time that PCT is expressed in PBMCs. This expression is modulated by bacterial LPS and sepsis-related cytokines. Therefore PBMCs may be among the sources of elevated PCT levels in patients with sepsis.

Functional role of CD26 on human B lymphocytes

CD26 is a well-known activation marker on T cells and natural killer (NK) cells [1]. It is identical with the ectopeptidase dipeptidyl peptidase IV (DP IV). The expression of CD26 on B cells has been discussed controversially [2,3]. We have studied the expression of this enzyme on B cells from the peripheral blood of healthy donors and of CVID patients, on cells of the Daudi Burkitt line and the EBV-transformed B-cell lines Jojo and Laz509. DP IV was detected by using anti-CD26 monoclonal antibodies and with help of specific enzyme substrates. Further the influence of specific synthetic DP IV inhibitors on mitogenic activation of purified B cells and DNA synthesis of cell lines was studied. We could show that in both groups 0-5% of freshly isolated CD20-positive B cells do express the CD26 antigen. After stimulation with pokeweed mitogen or St. aureus protein, the fraction of CD26-positive cells was enhanced up to 51% and 36%, respectively. Interestingly, induction of CD26 expression on B cells from CVID patients occurs in a manner similar to the B cells from healthy donors. Treatment of peripheral blood B cells and B-cell lines with highly specific competitive DP IV inhibitors leads to a significant inhibition of DNA synthesis in a dose-dependent manner. These data show that CD26 can be considered to be an activation marker not only of T- and NK cells but also of a main population of B cells, suggesting an involvement of CD26 in B-cell activation.

A detailed protocol for the measurement of TGF-β1 in human blood samples

Quantification of the multifunctional cytokine Transforming Growth Factor-beta1 (TGF-beta1) in blood samples has aroused increasing interest in recent years, since an abnormal regulation of this cytokine appears to play a key role in the pathogenesis of different diseases, such as autoimmundiseases or malignant tumors. The measurement of TGF-beta1 is complicated by a lot of problems concerning the collection, preparation and handling of blood samples, the platelet contamination, and the TGF-beta1 activation procedure. Here, we recommend detailed instructions for measurement of TGF-beta1 in blood plasma samples which should be followed to exclude the determination of false positive or negative results.

Increased Transforming Growth Factor β1 Plasma Level in Patients with Renal Cell Carcinoma:A Tumor-Specific Marker?

Purpose: The most worrying problem with renal cell carcinoma (RCC) seems to be the prediction of metastases by means of tumor-specific markers. Therefore, much effort is committed to the development of new markers. Materials and Methods: The level of latent transforming growth factor β1 (TGF-β1) was measured in plasma samples by ELISA. These samples were collected from patients with RCC before they underwent radical nephrectomy, from patients 1 h after extracorporeal lithotripsy, from patients with pyelonephritis, and from healthy controls. Results: In all cases of RCC the levels of latent TGF-β1 in plasma were much higher (n = 20, 41.0 ± 13.9 ng/ml, range 19.3–78.1 ng/ml) than in healthy controls (n = 20, 3.8 ± 2.9 ng/ml, range 0.6–9.9 ng/ml, p < 0.0001). The TGF-β1 levels in plasma after extracorporeal lithotripsy (n = 20, 7.4 ± 4.64 ng/ml, range 2.9–21.7 ng/ml, p < 0.01) and in patients suffering from pyelonephritis (n = 20, 18.93 ± 14.2 ng/ml, range 4.2–46.7 ng/ml, p < 0.001) were also higher than in healthy controls. Conclusion: We conclude that increased levels of latent TGF-β1 are common in the plasma of RCC patients. The TGF-β1 plasma level in RCC was found to be significantly higher than in cases of inflammation. Thus, TGF-β1 is a possible tumor-prognostic marker in RCC.

Interferon–Alpha Suppresses the Antiapoptotic Effect of NF–kB and Sensitizes Renal Cell Carcinoma Cells in vitro to Chemotherapeutic Drugs

Background: Immunochemotherapy (ICT) with interleukin–2 (IL–2) and interferon–α (IFNα) with a secondary effector (5–fluorouracil, 5 FU) is the only promising treatment for advanced renal cell carcinoma (RCC). With IFNα, besides the activation mechanisms of the immunosystem, a direct antitumor effect on tumor cells is expected. Materials and Methods: NF–kB activity in three permanent cell lines (Hep2, HepG2, HT29) and in primary RCC cell lines was measured after incubation with tumor necrosis factor–α (TNFα), IFNα, IFN–γ, TNFα+IFNα, and IFNγ+TNFα, respectively. NF–kB activity and induction of apoptosis by chemotherapeutic drugs (5FU and doxorubicin) were determined in cells transfected with a constitutively active NF–kB p65 or a dominant negative IkB. Results: NF–kB signaling induced by TNFα is suppressed by IFNα and IFNγ in the permanent cell lines and in the primary RCC tumor cell cultures. In an in vitro ICT model we show that pretreatment of RCC with IL–2 and IFNα leads to a diminished NF–kB response to TNFα. In certain tumors, this correlates with increased susceptibility to investigated chemotherapeutic drugs as shown by annexin stain and cell elimination. Modulation of the cellular NF–kB state by a constitutively active p65 or a dominant negative IkB mimics this effect. The IkB construct leads to the same effects as IL–2/IFNα pretreatment as shown by predominant elimination of the transfected cells from the overall population, while introduction of p65 leads to a partial rescue from the effect of IL–2 and IFNα. The described effect, however, applies only to a selection of primary cell cultures. Conclusions: Besides the immunomodulation effects, treatment of RCC with IL–2/IFNα leads to a proapoptotic state in certain tumors. The relevant mediator seems to be IFNα by suppression of the antiapoptotic effect of NF–kB. These data can provide an experimental base for correlation with real patient outcome after ICT.

Serum Transforming Growth Factor-beta 1 in Patients With Renal Cell Carcinoma

PURPOSE A major problem of renal cell carcinoma is the prediction of metastases via tumor prognostic markers. Therefore, much effort has been committed to the development of new prognostic markers. MATERIALS AND METHODS The level of transforming growth factor-beta1 was measured by enzyme-linked immunosorbent assay in serum samples collected from patients with renal cell carcinoma before radical nephrectomy. RESULTS In serum samples from 21 patients with renal cell carcinoma and 21 healthy controls mean transforming growth factor-beta1 levels were 177 +/- 54.1 versus 65.6 +/- 15.8 ng./ml., respectively. This difference was statistically significant (Mann-Whitney U test p <0.001). CONCLUSIONS Increased levels of transforming growth factor-beta1 are common in serum of patients with renal cell carcinoma.

Macrophages from rat livers with micronodular and macronodular cirrhosis differ with respect to mediator release and DNA-synthesis

BACKGROUND/AIMS Liver macrophages play an essential role in necro-inflammatory liver damage which leads to fibrosis and cirrhosis. The aim of the present study was to compare the mediator release and the DNA synthesis of macrophages at an early and at a later stage of liver cirrhosis induced by thioacetamide. METHODS Liver macrophages were isolated by an enzymic digestion method, followed by elutriation. The release of reactive oxygen species and cytokines, and the synthesis of DNA were measured in cultivated cells. RESULTS The vitality of isolated macrophages from cirrhotic livers was always higher than 98%. The total yield of macrophages was less in micronodular cirrhotic livers and was markedly higher in macronodular cirrhotic livers when compared with age-matched controls. The cellular granules measured by sideward light scattering showed a shift to larger sizes in macrophages from micronodular cirrhotic livers when compared with the controls and the other experimental group. Macrophages from both cirrhosis groups exhibited a markedly higher unstimulated and lipopolysaccharide-stimulated IL-6 production than the controls. The release of TNF-alpha did not differ between controls and the experimental groups. Macrophages from macronodular cirrhotic livers produced higher amounts of nitric oxide but less superoxide anion radicals than the controls. DNA synthesis was 10-12-fold and 3-10-fold higher in macrophages from micronodular and macronodular cirrhotic livers, respectively, when compared with the age-matched controls. CONCLUSIONS The data presented provide evidence that it is possible to isolate and to cultivate macrophages from livers with high yield and vitality at different stages of cirrhogenesis. Our results clearly demonstrate functional differences between macrophages from livers with micro- or macronodular cirrhosis; this finding may be important for the pathogenesis or perpetuation of the cirrhogenetic process.

Differential expression of cytokine genes in CD27-positive and -negative CD4 lymphocyte subsets from healthy humans and rheumatoid arthritis patients.

Immunofluorescence analysis of CD27 expression by CD4 lymphocytes from the peripheral blood of healthy humans or rheumatoid arthritis (RA) patients and from the synovial fluid (SF) of RA patients was carried out, along with the estimation of cytokine gene [interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10 and interferon-γ (IFN-γ)] expression in these lymphocyte subsets by RT-PCR. Although no differences in CD27-positive and negative peripheral blood CD4 cell subset distribution were revealed, marked differences in IL-3, IL-4, IL-5 and IFN-γ mRNA expression were detected between these lymphocyte subsets and between control and disease states. These results showed that phenotyping of different cell subsets in disease cannot provide adequate information about lymphocyte functional status. To estimate differences in cytokine gene expression, CD4 lymphocytes from the peripheral blood and SF of RA patients were compared. In both cases, mRNAs for IL-2, IL-4, IL-10 and IFN-γ were detected, but CD4 cells from SF failed to express detectable levels of IL-5 mRNA despite our findings of a CD27- cell accumulation within the synovial population of CD4 lymphocytes. These are the first data to demonstrate that expression of the IL-5 gene in RA SF CD27- lymphocytes is down-regulated and that IL-5 disregulation in RA cannot be ruled out.

Lymphocyte transformation test (LTT) – a model for testing the cytokine-induced immunomodulatory capacity of renal cell carcinoma

SummaryWe investigated the immunomodulatory capacity of cytokines produced by renal cell carcinoma in vitro by analyzing their effects on mitogen-induced T-lymphocyte blast cell transformation. All of the tested 70 cell cultures, derived from 70 tumor areas in 33 patients, had immunomodulatory capacity. In addition to suppression in the lymphocyte transformation test (max. 44/70; 63 %) there was also superinduction (max. 37/70; 53 %). We found no significant correlation with the stage and grade of primary tumors. However, the suppression of mitogen-induced T-lymphocyte blast cell transformation was significant in multifocal tumors (0.08 % TCM, P < 0.001) and non-significant in metastatic tumors. The production of the assayed cytokines IL-6, IL-10, IL-11, and TGF beta 1 was variable and there was no significant correlation to the immunomodulatory capacity of the tumors.ZusammenfassungZur Untersuchung von immunmodulatorischen Effekten der von Nierenzellkarzinomzellen in vitro abgegebenen Zytokine wurde im allogenen Lymphozytentransformationstest (LTT) der Einfluß von Nierenzellkarzinom-Kulturüberständen auf die mitogeninduzierte T-Lymphozytentransformation geprüft. Für alle untersuchten 70 Zellkulturen aus verschiedenen Tumorarealen von 33 Patienten konnte ein immunmodulatorischer Effekt nachgewiesen werden. Dabei kam neben der Hemmung im LTT (max. 44/70; 63 %, abhängig von der Konzentration) auch eine Aktivierung der T-Lymphozytentransformation vor (max. 37/70; 53 %). Der immunmodulatorische Effekt der Kulturüberstände der untersuchten Tumorareale zeigte keine signifikante Korrelation mit der lokalen Ausdehnung und dem Differenzierungsgrad des Primärtumors (T- bzw. G-Stadium). Bei multifokalem Tumorwachstum (12 Patienten) war die Hemmung im LTT jedoch statistisch hoch signifikant (0,08 % TCM, p < 0,001) verstärkt, bei metastasierenden Tumoren (5 Patienten) tendentiell. Für keines der von den Tumorzellen sehr variabel produzierten Zytokine IL-6; IL-10; IL-11 sowie TGF beta 1 ergab sich ein signifikanter Zusammenhang mit der immunmodulatorischen Potenz des Tumors.

Differential Expression of Cytokines and Cytokine Receptors in Renal Cell Carcinoma

Modulation of immunomechanisms by tumor cells can be caused by secretion of cytokines. In vitro data are usually gained in culture systems. It is debatable whether these systems are representative of the conditions inside the respective tumor tissues. Immunohistochemical studies of tumor tissue cryostats were compared with those in matched primary renal cell carcinoma tumor cell cultures. Results were correlated with histopathological characteristics and the in vitro cytotoxic effect of autologous tumor infiltrating lymphocytes (TILs). Expression of all studied cytokines and cytokine receptors could be shown in cryostats and cell cultures, but the detection pattern varied individually. The immunohistochemical results in cryostats were in good accordance with those in cell cultures. Expression of TGFβ1 both in cryostats and cell cultures significantly correlated with the lack of cytotoxic activity of autologous TILs. Representative data can be obtained in tumor culture systems of primary renal cell carcinoma. TGFβ1 secretion could play an important role in the interactions between tumor and cytotoxic cells.