Phytohemagglutinin mitogen

Substances that cause cells, particularly lymphocytes, to undergo cell division. Mitogens are also referred to as polyclonal activators, since they stimulate proliferation of lymphocytes irrespective of their clonal origin. The best known mitogens are phytohemagglutinines isolated from certain plants. 

The mitogenic action of phytohemagglutinin on resting peripheral leukocytes is best seen about 72 h after addition of the mitogen. However, significant increases in mitotic cells can be seen at 48 h. 

Fig. 12. Summary of sequential investigations carried out on a patient with Bence Jones myelomatosis. Note the high blood urea, high proteinuria, and the early elevated pokeweed mitogen (PWM) response instead of elevated phytohemagglutinin (PHA) response. Urine protein , blood urea O, PWM PHA ratio o, IgM,, IgA X,IgG.

The various organs of the immune system such as spleen, lymph nodes, thymus and bone marrow containing the cells involved in the various immune responses offer the possibility to harvest these cells and perform in vitro assays for evaluation of effects on the immune system. When part of an in vivo animal study this may indicate a direct toxic effect of pharmaceuticals, that is, immunosuppression (Table 18.2). So, it is feasible to obtain cell suspensions for further evaluation such as determination of cellular subsets of T and B leukocytes by fluorescent activated cell sorter analysis (FACS analysis), and determination of natural killer (NK) cell activity of the spleen cell population. An advantage of this approach is that it may lead to identification of a biomarker to be used in clinical studies. In addition, in vitro stimulation of spleen cells with mitogens activating specific subsets may indicate potential effects on the functionality of splenic cell populations. Concanavalin A (Con A) and phytohemagglutinin (PHA) activate Tcells, while lipopolysaccharide (LPS) activates primarily B cell populations. Blood is collected for total white blood cell (WBC) determination and blood cell differential count. In addition, serum can be obtained for determination of serum immunoglobulins. 

Ogihara. Effects of five Kampohozais on the mitogenic activity of li-popolysaccharide, concanavalin A, Phorbol Myristate acetate and phytohemagglutinin in vivo. J Ethnopharmacol 1986 18(2) 193-204. 

Dual staining of cells can also be used to look at DNA and protein markers simultaneously. Using methods similar to those described in the previous chapter for looking at cytoplasmic proteins simultaneously with surface membrane proteins, cells can be stained for surface proteins, then fixed and permeabilized, and then stained for DNA. Figure 8.16 shows the way that this type of technique can be used to permit the cell cycle analysis of subpopulations of cells independently. In this particular example, it can be seen that, after treatment with the mitogen phytohemagglutinin, it is the CD8-positive cells more than the CD8-negative cells that have been induced to enter S phase. 

In vitro lymphocyte proliferative responses to mitogens such as phytohemagglutinin, concanavalin A, and pokeweed mitogen or to specific antigens of interest. The response may be measured by tritiated thymidine incorporation or by cytokine production (IFN-r, IL-2, TNF, and others). 

Several processes in the immune response are affected by lithium in vivo and in vitro 139). The proliferative responses of hamster lymphoid cells to concanavalin A or phytohemagglutinin, which stimulate mitosis in T cells, were enhanced by lithium in a serum-free culture system. Proliferative stimulation also was obtained with lithium using the B cell mitogen lipopolysaccharide, but the B cell mitogens dextran sulfate and trypsin had no effect 140-143). Lithium increased the effects of suboptimal concentrations of stimulants, but had smaller effects on stimulation by optimal concentrations. With concanavalin A, the response to optimal stimulatory concentrations was inhibited 140). Paradoxical results such as these may be due to inhibitory effects of lithium on adenylate cyclase, or to effects on membrane transport systems 141). Most of these experiments used very high concentrations of lithium, considerably in excess of normal therapeutic doses (maximal inhibitory concentrations were 10 mM with hamster cells and 5 mM with human lymphocytes). At therapeutic levels of lithium, increased incorporation of [ H]thymidine was seen in human peripheral blood mononuclear cells. 

One reason to believe that substance P is an important immune effector molecule is that extraordinarily low concentrations are required for effects on lymphocytes. For example, substance P augmentation of the mitogenic effects of Con A or phytohemagglutinin M on human T cells is apparent at concentrations as low as 10" ° M (Payan etal., 1983). The ability of substance P to evoke the release of IL-2 from Con A-stimulated murine CD4 T cells is maximal at 10" M (Rameshwar etal., 1993). Substance P appears to bind to Nkl receptors on murine splenocytes the recognition of the C-terminus of the molecule by these receptors is indicated by the ability of SP4-11 to evoke the release of 11 2, whereas SPl-4 is inactive (Rameshvrar etal., 1993). In addition, substance P is much more active than either NkA or NkB, and the Nkl receptor ants nist CP96345 inhibits the release of IL-2 by substance P in a dose-dependent manner. 

A significant suppression of the lymphoproliferative response to T-cell mitogens, concanavalin A, and phytohemagglutinin was observed in male B6C3Fj mice administered 2.9 or 14.3 mg Hg/kg/day as mercuric chloride in drinking water for 7 weeks (Dieter et al. 1983). A significant decrease in the weight... 

This cytokine was discovered in the supernatants of T-ceU cultures that had been stimulated by antigens or mitogens, such as phytohemagglutinin (PHA). The recognition of IL-2 as a growth and proliferation factor for lymphocytes (namely T cells) made it possible to grow and expand infected T ceils for extended periods of time. This allowed the characterization of human T-lymphotfopic virus type 1 (HTLV-1) and HIV. ... 

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