October 8, 2008

The Role of Adherent Cells in the Immunosuppressed State of Mouse Progeny Transplacentally Exposed to Benzo([Alpha])Pyrene

By Urso, Paul Kramer, Mary K

Abstract In recent studies, we showed that murine fetal liver cells from progeny exposed to benzo(alpha)pyrene in utero by intraperitoneal injection of the dam at midpregnancy (12 d) suppressed cell proliferation in an allogeneic mixed lymphocyte response. On the other hand, fetal liver cells from the corn oil (vehicle for the carcinogen)-exposed progeny (control) appeared to enhance proliferation. Suppression or enhancement appeared to be mediated by fetal liver bearing CD8^sup +^ and Lyt 1^sup +^ (CD5^sup +^) cells. Despite these manifestations, the role of third-party cells needs to be considered. As a first premise, adherent cells were targeted as possible third-party cells. To test the role of the adherent cells, liver cells from benzo(alpha)pyrene-exposed fetuses were treated with ficoll-hypaque, and the interface cells were fractionated through glass wool or nylon wool. It is known that adhering cells, macrophages and B cells, readily attach to glass or nylon wool. The effluent cells and the adherent cells were cultured with syngeneic responder cells and allogeneic stimulator cells in a mixed lymphocyte response. The results showed that benzo(alpha)pyrene-effluent cells led to the enhancement of proliferation in the mixed lymphocyte response, while benzo(alpha)pyrene-adherent cells led to suppression. The effluent and adherent cells of com oil controls did not modify cell proliferation in the mixed lymphocyte response. These data suggest that a third-party cell, reasonably the macrophage or possibly B cells or both, since these are adherent cells, play a decided role in mediating suppression in the benzo(alpha)pyrene-exposed progeny. Keywords Adherent cells * Suppression * Benzo(alpha)pyrene * Mixed lymphocyte response


Mouse progeny transplacentally exposed to benzo(alpha)pyrene on the 12th d of gestation are immunodeficient for almost the entire life span (at least 24 mo), and about 9 mo after birth, there is a logarithmic increase in tumor frequency that reaches a peak of about 85%, 24 mo later (Urso and Gengozian 1984). During the course of their life, we found that the progeny demonstrate changes in T cell profiles involving depressed CD4^sup +^ CD8^sup +^ (double positive) T cells in the thymus, increased double negatives, deficient gammadelta, alphabeta T cell receptor molecules, presence of B(alpha)P-7,8-dihydrodiol-9,10-epoxide (BPDE) covalently bound to deoxyribonucleic acid (DNA) in the thymus, spleen and fetal liver cells, and dysfunctional T cell subsets (CD4^sup +^, Thy1^sup +^, CD5^sup +^) in supporting an allogeneic mixed lymphocyte response (MLR; Moolenaar-Wirsiy et al. 2007; Urso 2008; Urso et al. 2008, unpublished data). Recently, we obtained evidence that suggests the involvement of CD5^sup +^ cells in the immunosuppressive nature of the progeny (Urso 2008).

Polycyclic aromatic hydrocarbons (PAHs) are dangerous environmental pollutants that are strongly carcinogenic. The most studied PAH, as well as perhaps the strongest carcinogen, is benzo(alpha)pyrene [B(alpha)P], a ubiquitous environmental pollutant that is found in such sources as commercial (automobile exhausts; Grimmer and Pott 1983); domestic (cigarette smoke; Hecht 1999; Phillips 2002), and industry (foundry workers; Santella et al. 1993). When this carcinogen enters into a mammalian organism, it is metabolized by the P450 enzyme system to several carcinogenic metabolic intermediates that are immunosuppressive (Lee and Urso 2007). The metabolic progression of B(alpha)P toward carcinogenicity is a function of the BPDE-DNA complex (Luch 2005), and it seemed plausible that this complex is responsible for immunosuppression. However, we found that most of the intermediate metabolites of B(alpha)P are immunosuppressive (Lee and Urso 2007) and that BPDE- DNA is only partly involved in and not completely responsible for immunosuppression (Moolenaar-Wirsiy et al. 2007). Thus, we hypothesize that third-party entities play a decisive role in contributing to immunosuppression.

Although an attractive hypothesis for immunosuppression demonstrated by these animals is that the BPDE-DNA complex is solely responsible for immune deficiency, our data (Moolenaar-Wirsiy et al. 2007) do not support this hypothesis. Thus, we demonstrated that BPDE-DNA^sup -^ (negative) T cells from B(alpha)P-exposed progeny are severely deficient in mounting an allogeneic MLR, as are cells that are BPDE-DNA^sup +^ (positive; Moolenaar-Wirsiy et al. 2007). In addition, we have evidence that CD5^sup +^ liver cells from B(alpha)P-exposed fetuses provide an inhibitory effect on the enhanced proliferation in an allogeneic MLR caused by CD8^sup +^ fetal liver cells (Urso 2008). This suggests that CD5^sup +^ cells (B-1a cells?) may be integrally involved in immunosuppression induced by B(alpha)P. However, the idea that CD5^sup +^ cells may be involved in mediating suppression needs to be verified. We hypothesize that, perhaps, a third-party cell (or cells) plays a significant role in the immunosuppressive status of the B(alpha)P- exposed progeny. To test this hypothesis, we have fractionated liver cells of B (alpha)P-exposed fetuses by nylon wool or glass wool and assayed the influence of resulting adherent and effluent (nonadherent) cells on cell proliferation in the allogeneic MLR. In the experiments reported here, we found that nylon wool or glass wool adherent liver cells from B(alpha)P-exposed fetuses inhibit cell proliferation in the allogeneic MLR, while the effluent cells enhance cell proliferation. The results are detailed in this report.

Materials and Methods

Animals, preparation of fetal liver ceils, and glass wool/ nylon wool fractionation. Pregnant C3H/Anf mice (Cumberland View Farms, Clinton, TN; H-2^sup k/k^ hapiotype) or C3H/HeJ mice (Jackson Labs, Bar Harbor, ME; also H-2^sup k/k^) were used in this study. The animals were maintained in the animal quarters under strict control, as described previously (Moolenaar-Wirsiy et al. 2007), and according to the guidelines of The Animal Care Committee, The Morehouse School of Medicine.

Pregnant dams were killed on day 18 of gestation after an intraperitoneal injection of 150 [mu]g B(alpha)P/g body weight into the dam on the 12th d of pregnancy. Fetuses were removed, and the livers of four to six littermates were recovered and pooled; the organs were teased in 1 x phosphate-buffered saline containing 5% fetal calf serum (PBSF) with sterile fine forceps, and the cell suspension was filtered through a sterile nylon sleeve to remove large clumps of cells. Cells were counted in a hemocytometer, and the resulting cell suspension was layered upon a sodium diatrozoate (ficoll-hypaque, F-H) solution, v/v, specific gravity 1.089, at a cell concentration of 30 x 10^sup 6^/ml (total cells ~120 x 10^sup 6^), 4 ml layered over 4 ml F-H. The preparation was centrifuged at 3,500 rpm at 20[degrees]C for 30 min, and the buffy coat cells (interface) were removed and added to a 5-ml column of nylon wool (or glass wool; Novimed Uni-Sorb, Tel Aviv, Israel) at 120 x 10^sup 6^ cells in 2 ml PBSF. The procedure for fractionation on the wool columns was similar to that of Julius et al. (1973) and Handwerger and Schwartz (1974). Before the addition of the cells, the column was charged with PBSF. The incubation of the column containing cells was at 37[degrees]C for 60 min; subsequently, the effluent (nonadherent) cells were collected in PBSF. To recover the adherent cells, the compression of nylon wool or glass wool was done several times in the presence of PBSF, which results in greater than 90% recovery of the adherent cells. Recovered cells were made to volume in Peck and Bach's culture medium (PBM; Peck and Bach 1973) and counted, the viability was assessed by the trypan blue dye exclusion (TBDE) method, and cells were made to the appropriate volume in PBM and temporarily stored on ice until use. The recovery of cells amounted to ~40 x 10^sup 6^ for effluent (nonadherent) cells and ~50 x 10^sup 6^ for adherent cells, for a recovery of ~75% of the population added to the column, similar to that obtained by others fractionating mouse spleen cells (Folch et al. 1973; Cone et al. 1977; Bennett et al. 1981). Of the 40% effluent cells, according to Cone et al. (1977) for mouse spleen, 95% are purified T cells, and of adherent cells recovered, ~85% are B cells according to Bennett et al. (1981). Similar evaluations were applied for the preparation of glass wool columns and recovery of cells. For spleen cells, the yield of purified adherent cells was 30% to 58% (presumably macrophages; Folch et al. 1973). We assume that fetal liver, a hematopoietic organ, is similar to the mouse spleen, since the former organ is also strongly hematopoietic as is the mouse spleen. The viability of the fractionated cell preparations was invariably greater than 95% by the TBDE method.

The allogeneic MLR and influence of adherent and effluent cells on cell proliferation. The effect of wool effluent and adherent cells was tested on a standard MLR. The MLR is explained in detail elsewhere (Urso and Gengozian 1984). Briefly, C3H splenic responder (R) cells at 1 x 10^sup 6^ were cultured with 1 x 10^sup 6^ (Balb/ cxDBA/2) stimulator (S) spleen cells in PBM. The S cells were first inactivated with 1,000 rad of gamma-rays. The culture was incubated at 37[degrees]C under 5% CO2 in a humidified atmosphere. For a standard MLR culture, triplicate wells are pulsed with 0.5 [mu]Ci ^sup 3^H-thymidine (specific activity 6.7 mCi/mM-DuPont, Wilmington, DE) on day 4 of incubation, and cells were recovered 16 to 20 h later on glass fiber filters using a Skatron cell harvester; scintillation cocktail was added to the filters, and beta-dis integrations depicting cell proliferation in the cultures were assayed by a Beckman L5801 counter. Proliferation is expressed in counts per minute (CPM). In triplicate cultures, 0.25 and 0.5 x 10^sup 6^ adherent cells or effluent cells (in PBM) were cultured with R+S cells as outlined above. Four days after incubation, the cultures were pulsed with ^sup 3^H-thymidine, cells were harvested 16 to 20 h later, and proliferation was assayed as described above.

Statistics. Significant differences between the cultures were calculated by Student's t test. Results are considered significant at p


The effect of effluent cells from nylon wool fractionation on cell proliferation in the MLR. The effluent cells from B(alpha) P- exposed fetal liver fractionated through nylon wool enhanced cell proliferation 2.5-fold in the MLR relative to proliferation that occurred in a standard MLR (p

The effect of effluent fraction from glass wool and nylon wool on cell proliferation in the MLR. The effluent from fetal liver cells fractionated on glass wool was compared to the effluent cells from nylon wool fractionation. Most strikingly, the enhancement of proliferation in the MLR was strong by B(alpha)P-exposed fetal liver effluent cells recovered after glass wool fractionation (twofold greater) when compared to proliferation in a standard MLR (Fig. 2). Furthermore, the proliferation enhanced by the glass wool effluent cells was significantly greater than nylon wool effluent cells (p

The effect of adherent cells from nylon wool relative to effluent cells on proliferation in the MLR. B(alpha)P fetal liver cells recovered from nylon wool fibers (adherent cells) suppressed proliferation in the MLR when compared to proliferation that occurred in a standard MLR (p

In Fig. 4, the data show the suppressive action of B(alpha)P- adherent cells recovered from glass wool and nylon wool on proliferation in the MLR (p

Kinetics of proliferation in the MLR influenced by glass wool and nylon wool effluent cells. The MLR was assayed from days 3 to 5 of cultivation; that is, ^sup 3^H-thymidine was used to pulse cultures (in triplicate) on days 3,4, and 5. R+S cells were cultured with glass wool or nylon wool effluent cells (at 0.25 x 10^sup 6^ each), and proliferation was assayed as described above. There is a significant elevation of cell proliferation at each time interval assayed when B (alpha)P-effluent cells, whether from glass wool or nylon wool, were cultured with R+S cells, while no change in proliferation occurred with similar cells from controls (Fig. 5).


The results clearly show that the liver of B(alpha)P-exposed fetuses contain elements that suppress proliferation in a MLR glass wool and nylon wool fractionation permits retention of these elements on the fibers, and as a result of their capture (adherence), they are grossly reduced from the cell population that passes through the wool. According to other investigators who used nylon wool and glass wool columns to isolate adhering cells from murine spleen, nylon wool adherent cells are primarily B lymphocytes (Julius et al. 1973; Handwerger and Schwarte 1974), while cells captured on glass wool are mostly macrophages (Folch et al. 1973).

We did not microscopically observe the cells recovered from glass wool or nylon wool. However, in a recent report (Urso 2008), we assayed the phenotype of the fetal liver T cells and found that those of the corn oil control and B(alpha)Pexposed fetal liver expressed CD8^sup +^ and CD5^sup +^ cells. In this same report (Urso 2008), we also showed that the CD5^sup +^ cells from B(alpha)P fetal liver inhibited proliferation in the MLR, while similar cells from corn oil controls did not. On the other hand, fetal liver CD8^sup +^ cells from B(Cc)Ps or controls enhanced cell proliferation (Urso 2008). We are in the process of characterizing, in more detail, microscopic and phenotypic appearances of the effluent and adherent cells. However, as we have shown in this report, such fractionated cells do not behave as suppressor or enhancer elements in normal fetal liver. Our data strongly suggest that B(alpha)P damage leads to an integral change in cellular elements of fetal liver and the establishment of suppressor cells, a situation that can aptly explain the immunedeficient nature of progeny exposed to this carcinogen during fetogenesis. Since macrophages and B cells adhere to glass wool and nylon wool, respectively, the damaging intermediate molecules (that are immunosuppressive) and ultimate molecule of B(alpha)P metabolism (i.e., 3-OH-BP, B (alpha)P-7,8- dihydrodiol, BPDE-DNA adduct; Lee and Urso 2007) may target these cells in changing their profile that establishes them as suppressor cells. This seems feasible since macrophages and B cells have an extremely efficient P450 enzyme system that can metabolize the carcinogen into potent intermediary reactive molecules (Ng et al. 1998; van Grevenynghe et al. 2004). If this analysis is correct, this is the first indication that the carcinogen evokes the production of suppressor macrophages and B cells that could be responsible for the prolonged immunodeficiency expressed by these progeny.

Even though the stimulus is unknown, under certain conditions, macrophages and presumably B cells are induced to change into suppressor cells, which may also be the case after insult with B(alpha)P. Thus, Bacillus calumette guerin induces a cellular change into suppressor macrophages (Bennett et al. 1981); cold stress induces the appearance of suppressor macrophages (Kizaki et al. 1997); Rahim et al. (2005) have found that withdrawal from morphine from mice establishes macrophages and B-1 cells that are immunosuppressive. Since we have shown that nylon wool adherent cells are immunosuppressive (presumably B cells), it is also feasible that B(alpha)P insult leads to a change in B cells to render a population (B-1) immunosuppressive. Koide et al. (2002) showed that gamma-interferon changes mouse CD5^sup +^ Bl cells to a macrophage-like morphology. Therefore, it seems feasible to assign a B(alpha)P stimulus that would change, significantly, the profile of macrophages and B cells into suppressor cells. Recently, we found that CD5^sup +^ cells of B(alpha)P-exposed fetal liver considerably reduce the enhancing effect of normal fetal liver CD8^sup +^, CD8^sup +^ cells on cell proliferation in the MLR (Urso 2008).

From the experiments described herein, we cannot evaluate the cellular characteristic of effluent cells from glass wool or nylon wool fractionation that enhance cell proliferation in the MLR. However, as indicated above, we have evidence that CD8^sup +^ and CD8^sup +^ T cells from control fetal liver enhance cell proliferation (Urso 2008). From nylon wool, most of the adherent cells are believed to be B lymphocytes, while the effluent cells are considered to be a subpopulation of relatively pure (~95%) T cells (Cone et al. 1977). For glass wool, the adherent cells are primarily macrophages (Folch et al. 1973), and the effluent cells are primarily T cells. Although it is tempting to assign suppressor action to macrophages and B cells, this designation must await additional analysis regarding the functional characteristics of the adherent and effluent cells. Nevertheless, a case for macrophages as the suppressor population in the glass wool adherent cells is supported by findings that characterize macrophages as suppressor cells in various physiological associations (Bennett et al. 1981; Kizaki et al. 1997; Koide et al. 2002). Similarly, B cells are good candidates as the suppressor cells in the adherent population from nylon wool fractionation, since B-1 cells, trapped on the nylon wool, have been implicated as suppressor cells (Thompson et al. 1980; Berland and Wortis 2002). To support the suppressor B cells hypothesis, Singhal and Duwe (1975) showed suppressor action by murine splenic B cells on the plaque (antibody)-forming cell response against sheep red blood cells. Studies to shed further light on these phenomena are ongoing. An interesting question is: In the MLR cultures where enhancement of cell proliferation occurs, what is proliferating? Other than the enhancement of cell proliferation, the studies reported here cannot specify exactly which cells are responsible for the observed proliferation. Indeed, responder cells recognize the allogeneic antigens and therefore proliferate. This is a requisite for responder cells recognizing the antigens of allogenic stimulator cells in the MLR. The additional mitotic action, however, from these studies, cannot be ascribed to specific cells. It is possible that the added fetal liver cells can proliferate, but these cells are not equipped to recognize foreign antigens. Further, as we have shown in a recent paper, liver cells, whether from a B(alpha)P fetus or a control fetus, weakly, if at all, recognize the allogenic stimulator cells (Urso 2008). In the study by Urso (2008), in a standard allogenic MLR, R+S cells gave a mean CPM of 14 x 103+-1.3 (standard deviation; eight experiments), while fetal liver cultured with stimulator cells gave a mean CPM of 4.9+-0.7 (eight experiments). Indeed, the fetal liver effluent or adherent cells cultured with R cells only show proliferation less than (CPM from 5 to 10 x 10^sup 3^) responder cells that recognize the allogeneic antigens; the fetal liver fractions cultured with S cells show even lower CPM (see above). It is tempting to ascribe additional proliferation to further stimulus of responder cells. Additional experimentation is necessary for identifying specific elements involved in mitotic augmentation.


We have demonstrated that in the immunodeficient progeny exposed to B(alpha)P at midgestation, adherent cells from nylon wool or glass wool fractionation suppress cell proliferation in the MLR. Our data indicate that adherent cells from glass wool are more suppressive than those from nylon wool (Fig. 4) suggesting that macrophages are more suppressive than B cells. Effluent cells, on the other hand, enhance cell proliferation. The characteristics of the cells that induce suppression or enhancement are not revealed by these studies, but it is possible, if not probable, that the adherent suppressors are macrophages and/or B cells. In this study, we introduce the possibility that immunodeficiency of the B(alpha)P- exposed progeny is significantly due to the establishment of suppressor macrophages and/or suppressor B cells, which, in some way, contribute to the state of immunodeficiency in progeny exposed to B(alpha)P at midgestation.

Acknowledgments This work was supported by grant no, PCE-5053-G- 00-3062 from The US Agency for International Development and grant no. R815813 from the US Environmental Protection Agency. We thank Ms. Nicole Downing for editorial assistance and computer expertise.

Received: 5 November 2007 /Accepted: 10 April 2008 /Published online: 12 July 2008 / Editor: J. Denry Sato

(c) The Society for In Vitro Biology 2008


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P. Urso * M. K. Kramer

Department of Microbiology and Immunology,

The Morehouse School of Medicine,

720 Westview Drive,

Atlanta, GA 30310, USA

P. Urso (*)

371 Clubland Circle,

Conyers, GA 30094, USA

e-mail: [email protected]

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