Pathweb - Virtual Pathology Museum

Museo de patología virtual que contiene imágenes e información (etiología, epidemiología, descripción, referencias...) de enfermedades y órganos.

Cytokines in human breast cancer: IL-1alpha and IL-1beta expression.

We hypothesize that interleukin 1alpha (IL-1alpha) and interleukin 1beta (IL-1beta) are present and tumor cell associated in human breast cancer (HBC) specimens. To test our hypothesis: a) immunologic analysis was performed on HBC histologic sections for IL-1alpha (n=49) and IL-1beta (n=42) distribution; and b) homogenates of HBC tumors were analyzed for levels of IL-1alpha (n=82), IL-1beta (n=101) and interleukin 8 (IL-8) (n=103) expression. Immunohistochemical analysis demonstrated the presence of IL-1alpha and IL-1beta in tumor cells in patients with invasive cancer and ductal carcinoma in situ. Quantitative analysis confirmed the presence and positive correlation of IL-1alpha and IL-1beta to IL-8, a known angiogenic factor, in cancer specimens. These studies demonstrate that tumor-associated IL-1alpha+, IL-1beta are present in the tumor microenvironment and may play a pivotal role in regulating breast tumor growth and metastasis.

Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissue.

UNLABELLED: Recently, we demonstrated the presence of Interleukin-8 (IL-8) in human breast cancer (HBC) tissue. We hypothesize that the IL-8 receptors are present and play a role in tumor cell and vascular endothelial cell (VEC) activation (e.g. proliferation and angiogenesis). MATERIALS AND METHODS: Immunohistochemical analysis for IL-8 receptors (IL-8RA and IL-8RB) was performed on 43 malignant and 8 benign breast tissue samples. RESULTS: Tumor cells expressed IL-8RA and IL-8RB in all of the malignant specimens. Only 50% of the benign ductal epithelial cell (DEC) samples expressed these receptors. The majority of small vessel endothelial cells (SVEC) expressed IL-8RA and IL-8RB, while large vessel endothelial cells (LVEC) showed primarily IL-8RB expression. CONCLUSIONS: Our results demonstrate that tumor and VEC express the IL-8 receptors and likely play a role in regulating tumor and VEC activation which controls proliferation, angiogenesis and metastasis in HBC.

The effect of the synthetic lecithin analogue, dimethyl-DL-2, 3-distearolyoxypropyl-2'hydroxylethylammonium acetate, on cytolytic T lymphocyte (CTL) activity.

The effect of the synthetic lecithin analogue, dimethyl-DL-2, 3-distearolyoxypropyl-2'hydroxylethylammonium acetate, on CTL cytolytic activity was studied. The analogue significantly inhibits H-2b anti H-2d cytolytic T lymphocytes at concentrations which do not impair lymphocyte viability, protein synthesis, or RNA synthesis. At these concentrations the inhibition is reversible upon removing the analogue. Thus, the inhibition produced by analogue simply is not a result of analogue toxicity. At higher concentrations of the analogue, CTL inhibition is very pronounced; however at these higher concentrations there is evidence of non-specific toxicity of the analogue on CTL.

Membranolytic and nucleolytic activities of cytolytic T lymphocyte clones.

In an attempt to see if the nucleolytic and membranolytic activities of cytolytic T lymphocytes (CTL) were totally independent and could be expressed independently, we cloned CTL and determined their membranolytic and nucleolytic activities. If the two lytic mechanisms were completely independent and could be independently expressed by individual CTL, we anticipated that we would find CTL clones exhibiting only one or the other activity. Initial examination of membranolytic and nucleolytic activities in 99 newly established CTL clones revealed a poor correlation (r = 0.4) between the two activities. In addition, some clones expressed membenolytic activity without nucleolytic activity, and others, nucleolytic activity without membenolytic activity. The results suggest that CTL have 2 or more separate and independent mechanisms that lead either to the membranolytic or to the nucleolytic lesions in target cells.

The protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibits PMA-induced promiscuous cytolytic activity but not specific cytolytic activity by a cloned cytolytic T lymphocyte.

Phorbol 12-myristate 13-acetate (PMA) induces the cytolytic T lymphocyte (CTL) clone 4D (H-2b anti-H-2d) to promiscuously kill the inappropriate target EL-4 (H-2b). The protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibited the PMA-induced promiscuous lympholysis. The concentration of H-7 that inhibited PMA-induced lympholysis by 50% (IC50) was calculated to be 4 microM, which closely approximates the reported IC50 of H-7 of 6 microM for PKC activity in vitro. In striking contrast, specific cytolysis of appropriate P815 (H-2d) target cell by CTL clone 4D was not inhibited by concentrations of H-7 which inhibited PMA-induced promiscuous lympholysis. These results indicate that PMA-induced promiscuous lympholysis of inappropriate target cell is triggered via activation of PKC, whereas PKC activation is not obligatory in triggering CTL clone 4D to specifically kill appropriate target cells. Thus, these data suggest that cloned CTL have two or more triggering mechanisms than may initiate one or more cytolytic pathways.

Cytotoxic T lymphocyte (CTL)-mediated cytolysis proceeds in the absence of Na+/H+ antiport activity: regulation of cytosolic pH by the Na+/H+ antiport in a cloned CTL.

Cytotoxic T lymphocyte (CTL)-mediated cytolysis of specifically bound target cells (TC) is thought to be triggered by cross-linking the T-cell antigen receptor (TcR). Biochemical events associated with TcR cross-linking include increased intracellular calcium levels [Ca2+]i, hydrolysis of phosphatidylinositol (PI), and an increase in intracellular pH [pH]i. Whereas CTL-mediated cytolysis of some TC is calcium-dependent, and PI hydrolysis is speculated to trigger the CTL lethal hit via activation of PKC, little is known about changes in [pH]i relating to activation of the lethal hit stage. We report regulation of [pH]i in a cloned CTL by the electroneutral Na+/H+ antiport during activation with PMA and specific antigen-bearing TC. Furthermore, using 5-(N-methyl-N-isobutyl) amiloride (MIBA), a potent antiport inhibitor, we demonstrate that Na+/H+ exchange is not required for activation of CTL cytolytic activity.

Protein kinase C (PKC) inhibitors inhibit primary (1-0) but not secondary (1-1) or cloned cytotoxic T lymphocytes.

The isoquinolinesulfonamide PKC inhibitors H-7 and H-8 inhibit primary, in vivo generated cytotoxic T lymphocyte (CTL) activity by 50% at concentrations approximating their reported Ki values for PKC, 6 uM and 15 uM respectively. However, a greater than ten-fold higher concentration of H-7 (100 uM) is required to reduce secondary or clone 8F CTL-mediated cytotoxicity by 50%. At this concentration H-7 is also reported to inhibit calmodulin (CaM)-dependent enzymes. To distinguish between the effect of 100 uM H-7 on PKC versus CaM the napthalenesulfonamide CaM antagonist W-7 was investigated. W-7 inhibited primary, secondary and clone 8F CTL-mediated cytolysis by 50% near its reported Ki value for CaM-dependent kinase activity, 12 uM. We conclude that W-7 and 100 uM H-7 reduce cytolysis by inhibiting CaM-dependent reactions and not PKC. Thus, these findings indicate that primary killers require both PKC- and CaM-dependent activation pathways for lethal hit delivery, whereas highly lytic cultured CTL use only one pathway dependent upon CaM.

Wheat-germ agglutinin initiates monocytoid cell killing of non-antibody-coated erythrocytes.

Cells bearing alien surface antigens can be specifically destroyed by antibodies directed to the antigens and certain Fc receptor-bearing lymphoid or monocytoid cells through a killing mechanism known as antibody-dependent cellular cytotoxicity (ADCC). The present study was undertaken to see if lectins could substitute for specific antibody in ADCC and initiate lysis of non-antibody-coated erythrocytes. Human peripheral blood leucocytes and mouse PU-5 monocytoid cell lines were used as effector cells. Target-cell destruction was measured by the specific release of 51Cr. Our data indicated that Wheat-germ agglutinin (WGA), but not concanavalin A or soybean agglutinin, could activate killing of non-antibody-coated erythrocytes by human peripheral blood leucocytes and PU-5 cells. The membrane structure on the effector cell that was triggered by WGA directly related to N-acetyl-glucosamine and may be adjacent to, if not part of, the Fc receptor.

Local activation of the killing mechanism in PU5 antibody-dependent cell-mediated cytotoxicity effector cells.

This study was undertaken to determine whether ADCC effector cells expressed their killing mechanism globally throughout the entire cell surface or locally in a restricted area. Nonantibody-coated erythrocytes were bound to PU5 ADCC effector cells by either Con A or protein A-Ig bridges, and the effector cells with the adherent nonspecific erythrocyte target cells were mixed with specific antibody-coated erythrocytes. Our results indicated that the Con A or protein A-Ig-linked erythrocytes were not destroyed while the effector cell was in the process of killing specific antibody-coated erythrocytes. Therefore, the ADCC killing mechanisms of PU5 cells require activation by the interaction of Fc receptor with specific antibody-coated erythrocytes, and these killing mechanisms, once activated, are expressed in a locally restricted area of the effector cells rather than globally throughout the entire surfaces of the ADCC effector cell. Previously, we investigated the expression of the killing mechanism in cytolytic T lymphocytes (CTL) and found that alloimmune CTL in the process of killing specific target cells did not kill nonspecific targets linked to the CTL by either wheat germ agglutinin or immunoglobulin bridges. Thus, with respect to the surface membrane expression of lytic activity, CTL and the PU5 ADCC effector cells are similar. Our findings support the view that the various effector killing cells have a common cytolytic pathway and differ only by the recognition and triggering mechanisms.

Effect of phorbol esters on alloimmune cytolysis.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) profoundly affects cytolytic T-lymphocyte activity. Alloimmune C57BL/6 (H-2b anti-H-2d) cytolytic splenocytes treated with TPA, 0.3 to 3.0 ng/ml, killed specific P815 (H-2d) targets significantly better than did untreated controls as measured in 4-hr 51Cr release microcytotoxicity assay. Higher concentrations of TPA in the 30- to 100-ng/ml range significantly inhibited cytolytic function. The non-tumor-promoting analog, 4 alpha-phorbol-12,13-didecanoate, failed to affect killing at all doses tested. TPA-induced augmentation of cytolytic function requires an immunologically sensitized splenocyte population, since normal nonimmunized splenocytes treated with TPA did not kill target cells. Furthermore, treatment of splenocytes with anti-Thy 1,2 antibody and complement abrogated killing, indicating that T-lymphocytes mediate the killing. The TPA-induced effect does not require macrophage-like cells, since augmented killing occurred despite the removal of glass-adherent or iron-phagocytosing cells. Finally, the cytolytically augmented effector cells remain immunologically specific, since the nonspecific targets, syngeneic EL4 (H-2b) and third-party L929 (H-2k), are not killed. Thus, low levels of TPA augment the cytolytic ability of alloimmune T-lymphocytes against their specific target cells.

Alloimmune cytolytic T lymphocyte activity: triggering and expression of killing mechanisms in cytolytic T lymphocytes.

Nonspecific target cells were bound to alloimmune cytolytic T lymphocytes (CTL) by either wheat germ agglutinin or immunoglobulin and evidence of nonspecific target cell killing was sought by the 51Cr-release method. None was found. Thus, merely "gluing" CTL to target cells is not sufficient to effect target cell destruction. In order to determine if the killing mechanisms of CTL, once triggered, would kill adhering nonspecific target cells, CTL with adhering nonspecific target cells were mixed with specific targets. Under these conditions specific target cells were killed but the nonspecific target cells adhering to CTL by virtue of either wheat germ agglutinin or immunoglobulin bridges were not. Thus, when antigen-specific receptors on CTL interact with specific target cell antigens, killing mechanisms are activated in a local restricted manner rather than globally throughout the surface of the CTL.