Splenic cells

Noonan, F. R, De Fabo, E. C., and Morrison, H., Cis-urocanic acid, a product formed by UVB irradiation of the skin, initiates an antigen presentation defect in splenic cells in vivo, J. Invest. Dermatol. 90, 92-99, 1988. 

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. 

Polybrominated Diphenyl Ethers. Information regarding the immunosuppressive potential of PBDE mixtures is essentially limited to evidence from acute-duration oral studies in animals exposed to relatively high doses of pentaBDE. The plaque-forming splenic cell antibody response to injected sheep red blood cells was significantly reduced in mice exposed to 72 mg/kg/day pentaBDE for 14 days (Fowles et al. 

A continuation of this line of studies for 6 days to 23 weeks at 300 ppm showed continued decreases in numbers of mature B- and T-lymphocytes produced in the bone marrow, spleen, and thymus (Rozen and Snyder 1985). Abnormalities of humoral and cell-mediated immune responses following benzene exposure are presumably caused by a defect in the lymphoid stem cell precursors of both T- and B-lymphocytes. Bone marrow cellularity increased 3-fold, and the number of thymic T-cells increased 15-fold in benzene-exposed mice between the 6th and the 30th exposure. No corresponding increase in splenic cells was noted. The marked increase in the numbers of cells in bone marrow and thymus was interpreted by the authors to indicate a compensatory proliferation in these cell lines in response to... 

Fig. 1. Binding of protein L to human B cells and to mouse B cells expressing human Igs. a Human peripheral blood lymphoid cells were stained with FITC-labeled anti-human CD 19 monoclonal antibody and biotinylated protein L, followed by Cy-chrome-labeled streptavidin. b Splenic cells from transgenic mice expressing human Igs [46] were stained with FITC-labeled anti-mouse B220 monoclonal antibody and biotinylated protein L, followed by Cy-chrome-labeled streptavidin. All monoclonal antibodies and their corresponding isotype controls were purchased from BD PharMingen.

Sikorski EE, Burns LA, Stern ML, Luster MI and Munson AE (1991) Splenic cell targets in gallium arsenide-induced suppression of the primary antibody response. Toxicol Appl Pharmacol 110 129— 142. 

In the hybridoma technique (see Fig. 2), mice are inoculated with a specific protein, the desired antigenic target, over several months. The mouse is then sacrificed, and splenic lymphocytes are harvested. The murine splenic cells are coincubated and fused with immortaUzed human myeloma cells in vitro [32]. The fused cells grow into colonies that effectively serve as antibody-producing biologic factories. Each colony produces molecularly identical antibodies arising from the same original mouse lymphocyte—hence the term monoclonal. Antibodies are then tested for specificity or desired immune effect. 

In addition to carcinogenic effects, animal studies have shown the effects of benzene exposure on the immune system. Reid et al. showed a significant decrease in splenic cell proliferation in mice exposed to benzene for 14 days. Experimental animal studies also reported reduced circulating white blood cells, as well as changes in spleen morphology and weight in various experimental animal studies. These experimental animal studies further support the observation from 1913 by Wintemits and Hirschfelder that rabbits exposed to... 

A stimulatory effect was observed on the lysosomal and peroxidal activity of peritoneal macrophages and splenic cells after five days in vivo treatment of C57BL6 inbred mice with the ethanolic extract of the roots of . angustifolia [62, 63]. 

However, withaferin-A, which was the first active principle isolated from Withania somnifera and also constituted the first member of a new class of phytosteroids (the withanolides), produced immunosuppression. Thus, it inhibited adjuvant arthritis in rats, graft vs host reaction in chicken, xenograft vs host reaction in mice, and depleted murine splenic cells in vitro [38-41]. This effect has been attributed to the glucocorticoid-like structure of withaferin-A [39]. Withania somnifera also reduces acute phase reactants [8] as well as a2-macroglobulin during inflammation. 

Preliminary experiments in our laboratory have indicated a disturbance in the functional activity of splenic cells from pantothenic acid-deficient rats immunized with diphtheria toxoid. In studies conducted in collaboration with Dr. Abram Stavitsky it was shown that splenic cells from immunized pantothenic acid-deficient rats, in contrast to those from normal immunized rats, were unable to fabricate antibody when cultured in vitro or when passively transferred to normal rats. In co-operation with Dr. Cecile Leuchtenberger evidence was obtained that the mean deoxyribonucleic acid content of isolated splenic nuclei from immunized pantothenic acid-deficient rats was lower than that of comparable controls. Deoxyribonucleic acid was determined by microspectrophotometric analysis of the Feulgen reaction (Leuchtenberger et al, 1951). These results may be interpreted to mean that the deficiency interfered with the acceleration of cellular division which normally accompanies antibody production in the spleen. Since cellular division is always preceded by an increase in deoxyribonucleic acid content, the direct participation of pantothenic acid in deoxyribonucleic acid synthesis becomes an intriguing possibility. 

Splenic cells of pantothenic acid-deficient rats immunized with diphtheria toxoid contained considerably less antibody than cells from normal, immunized rats (Stavitsky et al., 1964). These results are further indication of the lack of any significant storage of preformed antibody in a deficiency state. 

Subgroup Type of recipient Source of splenic cells Number of mice grafted Number 0 tolerant mice... 

Figure 4. Growth Responses of Recipient CBA/J mice. Periods during which the Purified Control (Complete) and Pyridoxine-deficient Diets were Fed are Indicated by the Unbroken and Broken Lines, Respectively. DB Refers to Deoxypyridoxine, A Single Injection of 0.5 ml of a Suspension of Splenic Cells (0.8 to 1,0 X 10 Cells) was Administered Intraperitoneally at the Indicated Times. Used by Permission, from Axelrod and Trakatellis (1964). Copyright by Academic Press.

Finally, nanomaterials can be deposited ( settle ) in different organs, such as the liver, spleen, vasculature, heart and bone marrow [14, 35, 51, 84]. Most nanomaterials deposited in the liver were engulfed by Kupffer cells [51]. The deposition location appears to be affected by the nanomaterials surface coating for example, a PEG surface coating can prevent the uptake of nanoparticles by hepatic and splenic cells [90]. On occasion, for drug delivery purposes, an intentional surface coating is required. For example, the delivery of nanomaterials to the brain is extremely difficult, although a specific receptor, apolipoprotein, has been used to facilitate such translocation [16, 20, 91, 92]. 

Changes in the immune function depend on the organ, dose and duration of ozone exposure. Concanavalin A (Con A) stimulates the cell-mediated arm of the immune system. Con A reactivity was unaffected at days 4 or 7 of 0.7 ppm ozone exposure (20h/day) but increased by 1.5 and 3 fold after 14 and 28 days, respectively. In contrast, splenic cells of mice manifested decreased reactivity to T-cell mitogens in vitro (phytohemagglutinin, PHA, 38% decrease. Con A, 75 5 decrease) but not to a B-cell mitogen (lipopolysaccharide, LPSO or to alloantigens [294]. 

In rats, 7 days of exposure to 1 ppm ozone enhanced the reactivity of splenic cells to PHA, Con A and EPS [295]. Ozone inhalation gives also rise to nonspecific changes. Exposure to ozone at concentrations of 0.4 to 0.8 ppm can induce weight losses and/or Ihymic and splenic atrophy. A similar effect has been observed if steroids are injected into mice, i.e., ozone can produce steroid-mediated effects on lymphocytes. 

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