Timing Of Immune Assays For Detecting Developmental Immunotoxicity

Development of the immune system has been more studied and better defined in the mouse than any other species (see Chapter 1). With this in mind, many immune function tests utilized in adult mouse models are not valid in perinatal mice simply because of the immaturity of their immune systems. Therefore, an adult mouse immunotoxicity risk assessment screening battery must be modified to be age-appropriate, if it is to be used for detecting postnatal immune deficits following prenatal chemical exposure. Several NTP assays are not functional assays, e.g., splenic cellularity, splenic and thymic organ to body weight ratios, leukocyte counts, and splenic lymphocyte surface antigen expression. These assays may have utility for detecting developmental immunotoxicity at very early ages, or even prior to birth (Holladay and Luster 1996). The considerable information available in the mouse as to when different arms of the immune system become functional at an adult level adds strength to the mouse for immunotoxicity testing, and will need to be considered when employing NTP functional assays for developmental immunotoxicity screening. These functional assays include antibody production, cytotoxic T lymphocyte activity, delayed type hypersensitivity response, natural killer cell activity, T and B lymphocyte mitogen assays, and the mixed lymphocyte response.

The individual assay that afforded the highest predictive value (0.78) for immunotoxicity detection in adult mice was the antibody plaque forming cell (PFC) assay (Luster et al. 1992). This assay measures ability to produce specific antibody following challenge with allogeneic (sheep) red blood cells. The PFC assay has been used to demonstrate developmental immunotoxicity, for instance mice exposed to the nonsteroidal estrogen diethylstilbestrol (DES) during the first five days of life displayed inhibited PFC response to sheep red blood cells into adulthood (Kalland 1980). Exposure of mice to benzo[a]pyrene during gestation similarly resulted in a profound and persistently depressed PFC response in the progeny that was still present 18 months after birth (Urso and Johnson 1987). However, it is not certain how early in life the PFC assay or a PFC-like assay may be useful in mice for detecting immunotoxicity. B lymphocytes collected from fetal mouse livers acquire the capacity to produce plaques against allogeneic antigens at about days 16-18 of gestation. These animals are then capable of producing a heterogeneous or "adult-type" response to normal immunization between one and two weeks after birth (Goidl et al. 1976). Performing the traditional PFC assay in two week old mice is therefore possible; however, an adult-level antibody response is not seen until mice reach about 6 weeks of age and is required for maximal predictive value of the assay (Holladay and Blaylock 2002). Nonetheless, the demonstration of altered capacity of late-gestation fetal liver B lymphocytic cells to produce plaques against allogeneic antigens could prove to be a useful predictor of developmental immunotoxicity.

The cytotoxic T lymphocyte (CTL) assay is a measure of cell-mediated immunity that has been widely used in adult mice. This test has an estimated individual predictive value for immune suppression of 0.67 in mice, and, when codepressed with the PFC assay, was found to have 100% predictability for immunosuppression (Luster et al. 1992). One study is available that showed diminished CTL activity in 8-week old mice that were exposed 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from days 6-14 of gestation (Holladay et al. 1991). Beyond this report, there are limited additional data regarding the use of the CTL assay to detect developmental immunotoxicity, including how early this assay might first be useful. In vitro CTL responses to alloantigen can be detected as early as 4 to 6 days after birth in mice, but do not reach adult levels until between 11 to 20 days of age (Sarzotti et al. 1996). Thus, CTL assays that are performed prior to postnatal day 20 in mice may provide useful immunotoxicity testing information, but, at the least, will need to be compared to age-matched controls.

The delayed-type hypersensitivity (DTH) response is a second measure of cell mediated immunity that has proved useful for detecting long-lasting effects of developmental immunotoxicant exposure. For instance, depressed DTH was demonstrated in mice 101 days after birth following prenatal exposure to chlordane (Barnett et al. 1985). This assay again may be useful in prepubertal mice. Full expression of DTH requires antigen processing and presentation by bone marrow-derived CD1a+ epidermal Langerhans cells (LC). These LC begin to populate the mouse skin on day 19 of gestation, but are not fully functional until one week after birth (Elbe et al. 1989). Das et al. (1990) reported decreased DTH responses in very young mice at postnatal day 10, following maternal hexachlorocyclohexane exposure. It is not known if agents like TCDD or chlordane may similarly affect the DTH response in mice at similar early ages, or how such early depression of DTH might correlate to persistent effects on DTH caused by these agents.

Natural killer (NK) cell ability to lyse target cells also has a high predictive value (0.69) for risk of immunotoxicity in adult mice. Prolonged depression of NK cell activity as an indicator of developmental immunotoxicity has been demonstrated in mice exposed to low or moderate doses of steroidal and nonsteroidal estrogens during gestation (Kalland and Forsberg 1981; Lanier 1998). NK cells express the necessary receptors during development that control both positive and negative effector functions, including LY-49, NKR-P1 and CD94/NKG2 (Lanier 1998; Siva-kumar et al. 1999). Late in gestation, NK1.1+ lymphocytes collected from mouse fetal liver possess the ability to lyse MHC class I-deficient targets (Sivakumar et al. 1997, Toomey et al. 1998). These data suggest that NK cell activity in perinatal mice might be employed as an early indicator of developmental immunotoxicity.

Lymphocyte mitogenesis assays have a history of common use in immunotoxi-cologic studies and are part of the NTP testing battery. These include T lymphocyte proliferative ability in the presence of mitogens concanavalin A (ConA) and phyto-hemagglutinin A (PHA), and B lymphocyte mitogenesis in the presence of lipopolysaccharide (LPS). Lymphocyte proliferation after mitogen stimulation has also been applied to developmental immunotoxicology experiments, with altered proliferation reported in adult mice that were exposed in utero to immunotoxicants (Luster et al. 1979; Ways et al. 1980; Spyker-Cranmer et al. 1982). Similar use of lymphocyte proliferation assays in subadults is limited. In one study, offspring of pregnant mice exposed to hexachlorocyclohexane showed enhanced splenic T and B lymphocyte mitogenesis in response to ConA and LPS, respectively, at postnatal day 10 (Das et al. 1990).

The age at which lymphocyte proliferation assays can first be effectively utilized to detect immunotoxicity again has not been well characterized; however, several reports indicate that proliferative ability in mouse lymphocytes is acquired early in life. Fetal mouse thymocytes respond comparably to adult thymocytes, to the mito-gen PHA or in an MLR, by day 17 of gestation; and to the mitogen ConA at about 2 to 3 weeks postnatally (Mosier 1977). Mouse fetal liver B lymphocytes respond similarly to adult B lymphocytes to LPS stimulation beginning at about 14 to 16 days gestation (Goidl et al. 1976). These results suggest that T and B cell mitogenesis

Table 4.3 Immunotoxicants Producing Fetal Thymic Atrophy

Immunotoxicant

Exposure Regimen

Ref.

2,3,7,8-tetrachlorodibenzo-p-dioxin 10 mg/kg, GD 14 (TCDD)

3,3',4,4'-tetrachlorobiphenyl (TCB) diethylstilbestrol (DES) ethylene glycol monomethyl ether (EGME) benzo(a)pyrene 7,12-dimethylbenzanthracene (DBMA)

T2 mycotoxin (T2 toxin)

6-16 mg/kg, GD 12 3-8 mg/kg, gd 10-16 100-200 mg/kg, GD 14-17

Fine et al. 1989; Holladay et al. 1991 d'Argy et al. 1987 Holladay et al. 1993a Holladay et al. 1994

Holladay and Smith 1994 Holladay and Smith 1995

Source: Modified from Holladay SD and Luster MI. 1996. Alterations in fetal thymic and liver hematopoietic cells as indicators of exposure to developmental immunotoxicants. Environ Health Perspect 104 Suppl 4, 809-813.

assays have potential for use as indicators of immunotoxicity beginning in late gestation, with data again compared to age-matched controls.

The most widely used and technically most straightforward measurements of developmental immunotoxicity include fetal thymus and liver weights, and cell counts in these organs. Organ weight and cellularity data have generally not been considered to be among the most sensitive indicators of immune competence, with the exception of the relatively useful measurement of thymus/body weight ratio, which has an estimated predictive value for immunosuppression in adult mice of 0.68 (Luster et al. 1992). Table 4.3 summarizes studies where fetal thymic weight or cell counts were reduced after immunotoxicant exposure, showing the utility of this measurement.

Developmental immunotoxicity studies have also made use of flow cytometry to examine alterations in marker expression in fetal thymus and liver leukocytes. Fine et al. (1989) observed reduced numbers of progenitor T cells in fetal mouse liver after maternal TCDD exposure, detected as reduced expression of the marker terminal deoxyneucleotydal transferase (TdT) in fetal liver. The reduced liver TdT levels corresponded to fetal thymic atrophy. Holladay et al. (1991) and Blaylock et al. (1992) similarly reported fetal thymic atrophy in TCDD-exposed mice. These authors also noted inhibition of thymocyte maturation, detected by CD4 and CD8 marker expression. These combined effects, thymic hypocellularity and inhibited maturation, appeared to contribute to a relatively persistent diminishment in postnatal immune function (CTL activity) lasting until 8 weeks of age (Holladay et al. 1991). It may be noteworthy that lymphocyte phenotyping in adult mouse spleen has a high predictive value for immunosuppression (0.83) (Luster et al. 1992); however, no comparable data are available regarding the predictive strength for immunotoxicity detection of similar phenotyping in fetal thymus or liver. Table 4.4 and Table 4.5 show summary fetal thymocyte and liver marker expression data after maternal exposure to a variety of immunotoxicants.

Exposure of pregnant mice to pharmacologic doses of DES produced fetal liver and thymic effects that in many ways paralleled those reported for TCDD. These included reduced fetal liver TdT+ progenitor T cells, thymic atrophy, and inhibited

Table 4.4 Effect of Gestational Immunotoxicant Exposure on CD4+8+ (DP) and CD4-8- (DN) GD 18 Fetal Thymocytes

% Within Each Phenotype

Table 4.4 Effect of Gestational Immunotoxicant Exposure on CD4+8+ (DP) and CD4-8- (DN) GD 18 Fetal Thymocytes

% Within Each Phenotype

Exposure

Control

Chemical

Agent

Regimen

DP

DN

DP

DN

Ref.

TCDD

3 mg/kg, GD 6-14

69.1

21.1

43.2*

37.3*

Holladay et al. 1991

DES

8 mg/kg, GD 10-16

71.4

22.0

63.6*

29.2*

Holladay et al. 1993

EGME

200 mg/kg, GD 10-17

82.1

12.0

72.3*

19.9*

Holladay et al. 1994

B(a)P

150 mg/kg, GD 14-17

78.0

18.1

33.4*

62.0*

Holladay and Smith 1994

T2 toxin

1.5 mg/kg, GD 14-17

71.6

23.6

55.0*

40.2*

Holladay et al. 1993b

* Indicates numbers reported were different from control, p <0.05.

Source-. Modified from Holladay SD and Blaylock BL. 2002. The mouse as a model for developmental immunotoxicology. Human Exper Toxicol 21.525-532.

Table 4.5 Effect of Gestational Immunotoxicant Exposure on Fetal Liver Markers

Markers Examined

Table 4.5 Effect of Gestational Immunotoxicant Exposure on Fetal Liver Markers

Markers Examined

Agent

TdT

CD44

CD45

CD45R

Mac-1

Ref.

TCDD

+

nd

nd

nd

nd

Holladay et al. 1991

DES

+

nd

nd

nd

nd

Holladay et al. 1993

EGME

nd

+

+

+

-

Holladay et al. 1994

B(a)P

+

+

nd

+

nd

Holladay and Smith 1994

DMBA

nd

+

nd

+

+

Holladay and Smith 1995

T2 toxin

nd

+

nd

+

-

Holladay et al. 1993b

Note: + indicates the compound significantly changed expression of the marker; -indicates the compound did not affect the marker; nd indicates not determined.

Source. Modified from Holladay SD and Blaylock BL. 2002. The mouse as a model for developmental immunotoxicology. Human Exper Toxicol 21.525-532.

thymocyte maturation (Holladay et al. 1993a). Ways et al. (1980) had previously reported immunosuppression in female mice that were exposed neonatally to DES, in the form of prolonged diminishment of splenocyte immune responsiveness and inhibited graft-vs.-host responses. Kalland (1980) reported depressed antibody response in female mice following neonatal exposure to DES. More recently, Kar-puzoglu-Sahin et al. (2001) reported impaired interferon-gamma secretion at 1 to 1.5 years of age in mice exposed to a single dose of DES during development. The developmental immune lesions in fetal liver and thymus caused by DES may in part explain these postnatal observations of immune modulation.

Additional studies have made use of fetal thymus and liver to detect effects of developmental immunotoxicants in mice, with varying results. Prenatal exposure to T2 mycotoxin caused profound fetal thymic atrophy; however, in vitro studies demonstrated that fetal thymocyte viability increased, rather than decreased, after exposure to T2 toxin (Holladay et al. 1993b). The increased thymocyte viability appeared to be related to inhibited synthesis of endonuclease by thymocytes, required for apoptosis, while thymic atrophy was related to both inhibited thymocyte proliferation and targeting of progenitor cells in fetal liver. Similar fetal thymic antiproliferative and fetal liver progenitor effects were seen in mice exposed in utero to the PAH, 7,12-dimethylbenz[a]anthracene (DMBA), and corresponded to dose-dependent fetal thymic and liver hypocellularity (Holladay and Smith 1995). Another PAH, B(a)P, also caused depletion of fetal liver prolymphocytes, thymic cellular depletion, and inhibition of normal thymocyte maturation (Holladay and Smith 1994). The industrial solvent ethylene glycol monomethyl ether (EGME) caused fetal thymic atrophy without affecting thymocyte proliferation. Similar to TCDD, DES, T2 toxin and the two studied PAHs, the EGME-induced thymic atrophy appeared to at least in part be related to targeting of prolymphoid cells in the fetal liver (Holladay et al. 1994). For these reasons, Holladay and Luster (1996) concluded that altered marker expression in both fetal thymus and liver appears to be a highly sensitive indicator of gestational immunotoxicant exposure.

Getting Back Into Shape After The Pregnancy

Getting Back Into Shape After The Pregnancy

Once your pregnancy is over and done with, your baby is happily in your arms, and youre headed back home from the hospital, youll begin to realize that things have only just begun. Over the next few days, weeks, and months, youre going to increasingly notice that your entire life has changed in more ways than you could ever imagine.

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