Protein kinase c-dependent pathway is critical for the production of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6).

The authors hypothesized that certain PKC isoforms play an important role in the induction of pro-inflammatory cytokine (TNF-alpha, IL-1beta, IL-6) synthesis. To test this hypothesis, the cytosol-to-membrane translocation of select PKC isoforms with tested cytokine production in human monocytes cultured in vitro was correlated. It is reported that in monocytes treated with phorbol ester (PMA), translocation of PKC isoforms alpha, betaII, delta and epsilon precede cytokine synthesis. Moreover, specific inhibition of PKC translocation that occurs in the presence of Calphostin C is reflected in downstream events: lack of MAP kinases phosphorylation, loss of DNA binding ability by AP-1 transcription factor, and the reduction of pro-inflammatory cytokine synthesis. Thus, the cytosol-to-membrane translocation of PKC isoforms alpha, betaII, delta and epsilon with the subsequent activation of: (1) MAP kinases; and (2) AP-1 transcription factor, may represent critical steps in the induction of signalling cascade leading to TNF-alpha, IL-1beta, IL-6 synthesis in human monocytes.

Modified expression of c-Fos and c-Jun proteins and production of interleukin-1 beta in patients with rheumatoid arthritis.

OBJECTIVE: To examine the relationship between the expression of c-Fos and c-Jun proteins and the METHODS: The expression of c-Fos and c-Jun proteins and the production of IL-1 beta in the PBMC of 11 patients with active RA was determine by Western blots and ELISA techniques, respectively. RESULTS: The spontaneous expression of c-Fos protein and the production of IL-1 beta was higher in RA patients. Under LPS treatment, the PBMC of both RA patients and healthy subjects produced similar high levels of IL-1 beta without any significant changes in the expression of c-Fos and c-Jun proteins. By contrast, PMA-induced production of IL-1 beta was impaired in RA patients and was preceded by the disregulated expression of c-Fos and c-Jun proteins when compared with healthy donors. CONCLUSION: It can be postulated that in some RA patients the spontaneously high production of IL-1 beta may be associated with the up-regulated expression of c-Fos protein in PBMC. On the other hand the impairment of IL-1 beta production in RA induced by the PKC-dependent pathway, may be related to disturbances in c-Fos and c-Jun protein expression. This dysfunction seems to be compensated by some unknown mechanisms implicated in LPS signalling, which is known to involve not only the PKC-mediated pathway.

Recombinant tumor necrosis factor alpha inhibits growth of methylcholanthrene-induced sarcoma and enhances natural killer activity of tumor-infiltrating lymphocytes in aging rats.

The effect of recombinant human tumor necrosis factor alpha (rHuTNF-alpha) on the growth of immunogenic, methylcholanthrene-induced sarcoma (MC-Sa) and natural killer (NK) cell activity of tumor-infiltrating lymphocytes (TIL) in adult and aging rats was investigated. In both groups of animals the growth of transplantable MC-Sa was markedly and similarly inhibited by multiple intratumoral (i.t.) injections of rHuTNF-alpha. This effect was accompanied by stimulation of NK activity of tumor-infiltrating lymphocytes in adult as well as in aging rats. Studies in vitro demonstrated additionally that rHuTNF-alpha was a potent stimulator of NK but not of ADCC (antibody-dependent cellular cytotoxicity) activity of spleen lymphocytes from healthy animals. Our results indicate that the antitumor effect of TNF-alpha is comparable in adult and in aging rats bearing immunogenic MC-Sa. The inhibition of MC-Sa growth may be attributed not only to the TNF-alpha-induced necrosis of the neoplastic tissue but also to the in vivo stimulatory effect of this cytokine upon the NK-type function of lymphocytes infiltrating the tumor mass.

Functional relationship between high-affinity E receptor and CD2-11.3 epitope.

The expression of both high-affinity E receptor (EhR) to sheep erythrocytes and CD2-11.3 epitope on activated human T lymphocytes suggests that these two structures may be functionally identical or closely associated. Therefore the aim of the present work was to determine if the CD2-11.3 epitope is involved in the early E rosette formation. The ability of normal and phytohaemagglutin (PHA)-activated human T lymphocytes to express the CD2-11.3 epitope and to form early E rosettes (T cells with EhR) was studied simultaneously. The partial divergence of CD2-11.3 expression on T lymphocytes from the ability of these cells to form early E (Ee) rosettes was found. The results indicated that the expression of CD2-11.3 epitope alone is insufficient to form the Ee rosettes by activated T lymphocytes, yet it may facilitate this phenomenon in the presence of EhR. The above data clearly show that the CD2-11.3 epitope is functionally closely associated although not identical to EhR. Accordingly, it seems that these two structures may co-adhere to the appropriate ligand. Thus it is possible that the CD2-11.3 epitope, as well as its established role in activation signalling, may also act as a co-adhesion molecule.

Methylcholanthrene-induced tumors in adult and in aging rats: infiltrating cells and their ADCC activity.

In our previous studies we found that the slower growth of syngeneic, immunogenic MC-induced sarcoma (MC-Sa) in aging rats was followed by the higher activity of spleen lymphocytes in ADCC assay. The purpose of the present paper was to study infiltrating cells of immunogenic and non-immunogenic MC-Sa and to estimate in situ ADCC activity in relation to the growth of MC-Sa transplants in adult and aging rats. It was found that MC-Sa's were infiltrated mainly by lymphocytes. Among cells infiltrating tumor the high percentage of cells with Fc receptor was present. During progressive tumor growth the percentage of infiltrating cells and also FcR+ cells significantly decreased within immunogenic MC-Sa, but did not change in the case of the non-immunogenic MC-Sa. In cell infiltrates of both tumors no differences between adult and aging rats were observed. At early stages of the tumor growth the cells active in ADCC assay were present within both MC-Sa. The slower growth of immunogenic MC-Sa in aging rats compared with adults was connected with longer maintenance of ADCC activity in situ. In the case of non-immunogenic MC-Sa, which grew at similar rate in both groups of rats, no differences in ADCC activity between adult and aging animals were observed. It is suggested that in situ ADCC activity may be one of the mechanisms responsible for the slower growth of immunogenic MC-Sa in aging rats.

Surface markers on human activated T lymphocytes. IV. Comparison of high-affinity E-rosette receptor expression with the expression of other activation markers (receptor for interleukin 2, MHC class II (antigens).

In order to re-examine the value of high-affinity E rosette receptor (Eh-R) as an activation marker of human T lymphocytes, its existence on resting and activated T cells was compared with the expression of such known activation markers as receptor for interleukin 2 (IL-2R; Tac antigen) and MHC class II antigens (DR/DP and DQ). To this aim expression of the above surface markers on lymphocytes of TEe subset, derived from early E rosettes (Eh-R+) and on lymphocytes of TEl subset, derived from late E rosettes (Eh-R-), was examined immediately after purification of the cells from peripheral blood as well as during cell activation with PHA. The phenotypic studies were done by using monoclonal antibodies and indirect immunofluorescence technique. We confirmed previous observation that in the course of PHA stimulation Eh-R like IL-2R marked currently activated T cells. However, it was also found that in the long-term cultures of lymphocytes activated with PHA, the expression of Eh-R was sustained on the cells which lost their IL-2R and DR/DP antigens. The above findings and the fact that TEe cell subset consisting of Eh-R+ lymphocytes was almost completely depleted from cells bearing IL-2R and MHC class II antigens allowed us to conclude that this subset of peripheral blood T lymphocytes represented not currently activated cells but the cells which had been previously activated in vivo.

Surface markers on human activated T lymphocytes. III. High-affinity E-rosette receptors.

High-affinity E-rosette receptor (EhR) is suggested to be an activation marker of human T lymphocytes. The present study has been undertaken to estimate the dependence of EhR expression upon the T cell activation. To this aim the expression of EhR on T cells stimulated with PHA was examined. Nonsynchronized and arrested in G1 phase of cell cycle cultures of T lymphocytes were employed. The kinetics of expression of either de novo induced or reexpressed EhR was estimated by using two T cell subsets (originally EhR- and EhR+). In accordance with opinion of others, our results have shown that EhR is an activation marker of human T cells. Moreover, we have found that EhR is the marker of early and late activation stage because: its expression is induced in G1 phase of cell cycle, it continues to increase with the time of mitogen stimulation and the maximal level of EhR expression coincides with the time of the maximal RNA and DNA synthesis. Both induced de novo and reexpressed Eh receptors display the same expression kinetics.

FcR+ lymphocytes and ADCC in tumor-bearing adult and aging rats.

It was shown that in the normal, aging rats as compared with adults a decrease of cytotoxic activity of spleen lymphocytes in syngeneic ADCC test appeared which was followed by the parallel decrease of percentage of EA rosetting lymphocytes. In adult rats with the syngeneic transplants of immunogenic, methylcholanthrene-induced sarcoma (MC-Sa) a dramatic decrease of ADCC and much smaller decrease of EA rosettes were observed in comparison with the normal animals. On the contrary, in aging tumor-bearing rats an increase of ADCC activity was found, however, it was not followed by the changes of EA rosettes. Moreover, it was found that the non-forming EA rosette lymphocytes demonstrated a slight ADCC activity mostly expressed in aging rats with MC-Sa.

ADCC in aging rats with methylcholanthrene-induced sarcoma (MC-Sa).

Investigations on the relationship between the aging of the immune system and the tumor growth are the trigger of our studies. The purpose of the present paper was to determine a relation between cytotoxic activity of the spleen lymphocytes in ADCC assay and the MC-induced sarcoma growth in adult and aging rats. In ADCC assay the mouse leukemia L1210 labeled with Cr and sensitized with rabbit anti-L1210 serum was used as target cells. In aging rats with MC-Sa tumors the lymphocyte activity in ADCC was increased or remained unchanged in the comparison with normal animals. On the contrary, in adult rats with MC-Sa tumors ADCC activity was decreased. In comparative studies between the groups of adult and aging rats a reverse relationship between the tumor growth and the lymphocyte activity in ADCC was found. In aging rats the level of ADCC was higher than in adults, but the tumor growth was slower. We suggest that ADCC phenomenon may be involved in an antitumor response, especially effective in aging rats.

Activity of lymphocytes and antibodies derived from aging and adult rats bearing a MC-induced tumor in the syngeneic ADCC test.

The purpose of the present paper was to demonstrate the antitumor activity of lymphocytes and antibodies in the antibody-dependent cell-mediated cytotoxicity test (ADCC) in relation to the tumor growth in adult and aging rats bearing a methylcholanthrene-induced sarcoma (MC-Sa). In the syngeneic system using the ADCC test effector spleen lymphocytes, 51Cr labeled MC-Sa target cells and anti-MC-Sa serum were derived from inbred male Wistar rats. It was found that adult and aging tumor-bearing rats possessed lymphocytes (LSa) and antibodies (Ab) which were active in ADCC. In comparative studies between the groups of adult and aging rats a reverse relationship between tumor growth and activity of LSa and Ab in the ADCC test has been found. Thus, the larger weight of tumors in the group of adult rats was followed by lower LSa and Ab activities in the ADCC test. On the other hand, the smaller tumor growth in the group of aging rats was followed by higher activities of LSa and Ab in this test. We suggest that the ADCC phenomenon could be one of the antitumor mechanisms especially effective in aging rats.

Regulatory role of lysosomal enzymes in rat lymphocyte reactivity in vitro.

The influence of three lysosomal enzymes, beta-glucuronidase, acid phosphatase and hialuronidase, the representatives of lysosomal enzymes which can be released by exocytosis, was tested o reactivity of rat lymph node lymphocytes in vitro. These three enzymes do not change the spontaneous lymphocyte stimulation. However, beta-glucuronidase enhances the lymphocyte response to PHA. On the contrary, acid phosphatase and hialuronidase suppress PHA-induced stimulation of lymphocytes. In the mixed lymphocyte cultures (MLC) beta-glucuronidase enhances or suppresses DNA synthesis depending on the degree of lymphocyte stimulation in control MLC. Acid phosphatase and hialuronidase suppress lymphocyte stimulation in MLC.

Regulatory role of humoral factors released by macrophages in rat lymphocyte reactivity in vitro. I. Influence of humoral factors released by sensitized peritoneal macrophages on normal lymphocyte reactivity.

Supernatants harvested from cultures of peritoneal macrophages, derived from rats sensitized to bacillary antigens exhibited an intense stimulating effect on syngeneic normal lymph node lymphocytes. Lymphocytes stimulated in this manner showed non-specific cytotoxic activity.

Regulatory role of humoral factors released by macrophages in rat lymphocyte reactivity in vitro. II. Influence of humoral factors released by normal peritoneal macrophages on spontaneous lymphocyte stimulation.

Supernatants harvested from peritoneal macrophage cultures, derived from normal rats, exhibit in vitro two antagonistic activities: stimulating and suppressing spontaneous stimulation of syngeneic lymphocytes. Stimulation of DNA synthesis by macrophage supernatants occurs in lymph node lymphocytes, which show low values of spontaneous stimulation. Suppression of DNA synthesis by macrophage supernatants is revealed in thymocytes and cortisone-resistant thymocytes, which demonstrate high values of spontaneous stimulation.

Regulatory role of humoral factors released by macrophages in reactivity of rat lymphocytes in vitro. III. Humoral factors released by lymph node macrophages regulating lymphocyte response to PHA.

Lymph node macrophages, derived from normal rats, generated in vitro factors enhancing or suppressing syngeneic lymph node lymphocyte stimulation in response to PHA and reconstituting the PHA-dependent response of lymphocytes deprived of native macrophages. The occurrence of one of the opposite activities depended on the degree of lymphocyte stimulation by PHA. Moreover, the expression of suppressing or stimulating effect no lymphocyte response to PHA of the same supernatants, changed twice with their dilution, (suppression leads to enhancement leads to suppression) suggesting the existence of two distinct factors: suppressing and enhancing.

Immunogenicity of methylcholanthrene-induced tumors tested by draining lymph node assay in syngeneic rats.

The immunogenic properties of MC-induced and not metastasizing tumors were tested in inbred Wistar and August strains by the aid of lymphocyte stimulation in syngeneic draining lymph nodes in the comparison with the opposite ones. The lymphocyte stimulation was estimated in vitro by the spontaneous incorporation of 3HTdR. It was found that the peak of the first response of draining lymph nodes was noted on the 7th day after injection of syngeneic tumor cells. This antitumor response was specific because: 1. the second immunological response of draining lymph nodes to the same tumor cells developed earlier and was higher than the first response and 2. the rats previously immunized with MC-induced tumor, and then challenged with syngeneic sarcoma or syngeneic fibroblasts exhibited the lack of the second response and only some cross-reactivity was observed by the use of the other MC-induced sarcoma. MC-induced tumors appeared earlier and more frequently in August than in Wistar strain, the level of syngeneic draining lymph node response to immunogenic tumors was higher in August than in Wistar rats despite of the lack of difference between the percentages of immunogenic tumors arising in these two rat strains.