Sodium caseinate induces secretion of macrophage colony-stimulating factor from neutrophils.

In this work we provide evidence that granulocytes produce macrophage colony-stimulating factor (M-CSF) in the band cell stage and secrete it upon sodium caseinate-mediated differentiation to polymorphonuclear cells. We identified M-CSF in an enriched population of myeloid band cells from murine bone marrow using a chromophore-labeled monoclonal anti-M-CSF antibody. An ELISA assay was then used to detect secreted M-CSF in culture supernatants of enriched band cells differentiated to mature neutrophils using sodium caseinate. Colony formation in vitro by the supernatants from differentiating band cells was blocked by anti-M-CSF, thus suggesting that this factor is the only one responsible for this activity. Our data imply that casein can modulate hematopoiesis possibly via M-CSF production. Finally we discuss the possibility whether this M-CSF in concert with G-CSF could establish a cellular communication network between macrophages and granulocytes allowing them to simultaneously arrive at the inflammatory site.

Alpha-, beta- and kappa-caseins inhibit the proliferation of the myeloid cell lines 32D cl3 and WEHI-3 and exhibit different differentiation properties.

We have recently shown that sodium caseinate (CasNa) was able to inhibit the proliferation of the myeloid cell line 32D cl3 in a non-toxic way, and that it also induced the expression of macrophage colony-stimulating factor (M-CSF). Casein is the main protein present in milk and is composed of alpha (alpha), beta (beta) and kappa (kappa) subunits. This work was undertaken to evaluate if any one casein is responsible for the proliferation and differentiation properties found for CasNa on myeloid cells. Taking into consideration that 32D cl3 cells are considered to be non-malignant and dependent on IL-3 for proliferation, we also included for this study a leukemic cell line, WEHI-3, that does not depend on any external growth factor for its proliferation in order to evaluate if the growth inhibitory effect of caseins is also present for malignant cells. Our results showed that all caseins were inhibitory for the proliferation of either 32D cl3 and WEHI-3 and that only the 32D cl3 cells were induced to differentiate into the monocyte-macrophage lineage. In order to evaluate if CasNa was able to inhibit the proliferation of other myeloid cells we used J774 and P388 and found that they were also inhibited. We also determined that the different caseins exhibit different differentiation properties, with alpha-casein being the only one able to induce the secretion of M-CSF. We consider this work to open a new field of research, where casein, or its components, can be studied for their possible role in hematopoiesis and on the inhibition of malignant cell proliferation for therapeutic use.

Thrombin increases hyposmotic taurine efflux and accelerates ICI-swell and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation.

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

Tyrosine kinases and osmolyte fluxes during hyposmotic swelling.

Recent evidence documents the involvement of protein tyrosine kinases (TK) in the signalling network activated by hyposmotic swelling and regulatory volume decrease. Both receptor type and cytosolic TK participate as signalling elements in the variety of cell adaptive responses to volume changes, which include adhesion reactions, reorganization of the cytoskeleton, temporal deformation/remodelling of the membrane and stress-detecting mechanisms. The present review refers to the influence of TK on the activation/operation of the osmolyte efflux pathways, ultimately leading to cell volume recovery, i.e. the osmosensitive Cl- channel (Cl-swell), the K+ channels activated by swelling in the different cell types and the taurine efflux pathway as representative of the organic osmolyte pathway.

Epidermal growth factor receptor is a common element in the signaling pathways activated by cell volume changes in isosmotic, hyposmotic or hyperosmotic conditions.

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume.

Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.

Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation.