T-cell homeostasis in mice exposed to airborne xenobiotics

Abstract

Many effects of environmental toxic agents contribute to the deregulation of immune system homeostasis. Here we demonstrate that the effect of airborne suspended matter (ASM) on the generation of mouse T cells is reversible. This reversal can be achieved by an active process that returns the T cells to homeostasis and does not result from the simple effect of ASM deprivation. An accelerated development of thymocytes and increased influx of T-cell progenitors to the thymus in mice exposed to environmental xenobiotics has been postulated. This hypothesis has been confirmed by parallel increases in the percentages of single-positive and triple-negative thymocytes. Enhanced expression of thymocyte surface markers related to positive selection has also been observed. The pathway of T-cell progenitor development is favoured in the bone marrow of mice exposed to ASM.

Introduction

Exposure to environmental xenobiotics causes multidirectional changes in various human and animal tissues and organs. These xenobiotics exert strong harmful effects on the immune system in susceptible individuals, resulting in diseases of enhanced or depressed activity (hypersensitivity/autoimmunity or immunosuppression/immunodeficiency, respectively).^{1, 2} The biological activity of atmospheric industrial xenobiotics on the vertebrate immune system is very complex and depends on various factors such as time and route of exposure, development stage of the immune system, dose, species, sex and age.^{3, 4} Normally, individuals are exposed not to a single chemical, but to a mixture. The final effect on the immune system activity results from the antagonistic, synergistic, or redundant effects of particular components. The airborne suspended matter (ASM) varies in composition and origin. The health risk is mainly associated with particles of 10 μm or less.^5–7

In a previous study, we investigated the immunotoxicity of ASM, consisting of carbonaceous particles that had adsorbed a wide spectrum of heavy metals (lead, copper, iron, chromium, cobalt, cadmium, manganese, nickel and zinc) and that had been collected from the highly polluted sites of urban agglomerate in Upper Silesia (Poland). We demonstrated the decrease of the percentage of double-positive (DP; CD4⁺ CD8⁺) thymocytes in mice exposed to an airborne chemical mixture, and we considered this to be a consequence of the high potential toxicity of ASM in extrapulmonary lymphoid organs.⁸ We postulated that such profound depletion of these cells could be explained by the increase of ASM cytotoxicity for immature thymocytes, or alternatively by an accelerated exportation of DP thymocytes to the periphery without cell destruction. We decided to study the mechanism of these changes. We demonstrated that the acute exposure of young mice to ASM caused a decrease in the percentage of thymocytes with intracellular interleukin-2 (IL-2) and IL-4, which was accompanied by a simultaneous increase in the secretion by these cells of both these cytokines into the medium.⁹ This may suggest different sensitivity of mouse thymocytes to toxic environmental xenobiotics, and may account for the possibility of rescuing thymocytes from apoptosis by elevated synthesis of IL-2 and IL-4.^{10, 11} Next, we showed that the proliferation response of thymocytes to the principal growth-promoting factors IL-2, IL-4 and IL-7 was increased after ASM exposure.¹² This may suggest that homeostasis-driven proliferation (HDP) contributes to the compensation of the loss of cells resulting from the effect of environmental xenobiotics. It has been shown in many systems that homeostatic proliferation is cytokine-dependent, and IL-7 seems to be crucial in this process.¹³ IL-7 and IL-4 can promote the proliferation and prolong the survival of thymocytes.¹⁴ IL-2 is thought to be involved in thymocyte selection and in the development of thymic stromal cells that is required for normal thymocyte differentiation.¹⁵ Increase of the percentages of CD3^high and T-cell receptor-β (TCRβ)^high thymocytes in mice exposed to ASM has been demonstrated.⁴ Both markers are commonly used to characterize the maturation process in the thymus.^{16, 17} These published results suggest that the immune system can evolve mechanisms responsible for protection against environmental xenobiotics. In this study we demonstrate that the reversibility of the effects of experimental exposure of mice to ASM is the result of an active process responsible for the restoration of T-cell homeostasis. The aim of our study was to confirm the hypothesis depicted above. The schedule of our experiments aimed to answer the following questions:

1. Are the changes in the distribution of thymocyte populations observed after exposure of mice to environmental xenobiotics reversible?

2. Is the apoptosis involved in the changes of the percentage of mature thymocytes (CD4⁺ CD8^– and CD4^– CD8⁺) and less mature (CD4⁺ CD8⁺) thymocytes?

3. Is the increase of the percentage of mature thymocytes the result of the acceleration of thymocyte development and the compensative influx of T-cell progenitors from the bone marrow?

Materials and methods

Mice

Female BALB/c mice (6 weeks old) purchased from the National Institute of Hygiene (Warsaw, Poland) and bred in our facility were used in all experiments. The animals were maintained at 22–24° under a 12-hr photoperiod, with food and water ad libitum.

Airborne suspended matter

ASM was collected at several locations within the industrial region of Upper Silesia (Poland) for use in these experiments. Particulates (size range 0·3–10 μm diameter) were removed from filters, suspended in phosphate-buffered saline (PBS) and sterilized. The Sanitary-Epidemiological Station at Katowice (Poland) provided chemical analysis of the samples. Heavy metals such as lead, copper, iron, chromium, cobalt, cadmium, manganese, nickel and zinc were identified as the principal chemicals adsorbed on ASM.

Animal exposure to ASM

Mice were exposed to ASM according to the acute model of toxicity. Animals were injected intraperitoneally with a single dose of ASM in 0·5 ml PBS corresponding to 170 mg ASM/kg of body weight. The dosage was chosen according to our previous studies and did not damage the lymphoid organs. The control mice (Ctrl) were injected with the same volume of PBS. The animals were killed 72 hr after ASM administration, and thymus, spleen and bone marrow were removed for further examination.

Cell preparation

Thymuses and spleens were isolated, homogenized and filtered to remove tissue debris. Erythrocytes in spleen suspensions were lysed with a lysing buffer that did not affect splenocyte viability. Bone marrow cells were isolated from the femurs and tibias by washing the bones with 5 ml PBS.

Surface marker staining

Monoclonal antibodies (mAb; Becton-Dickinson, San Diego, CA) coupled with diverse fluorochromes were used for direct staining of the cells. Cells for staining were suspended in PBS + 0·5% bovine serum albumin + 0·01% NaN₃. For the examination of triple-negative (TN) thymocytes (CD3^– CD4^– CD8^–), cells were stained with mAb against CD3FITC/CD4PE/CD8PE. Monoclonal antibodies a-CD4PE and a-CD8FITC were used to examine CD4^low thymocytes. The expression of CD132 was analysed in thymocyte populations that differed in their TCRβ expression. The mAbs a-Thy-1.2APC and c-KitFITC were used as markers of early thymocyte development. Bone marrow cells were stained according to the following schedule: CD127PerCP/c-KitFITC/Sca-1PE to evaluate the early phase of lymphoid cell development; CD127PerCP/Sca-1PE/Thy-1.2APC to determine the early phase of T-cell development; and CD127PerCP/Thy-1.2APC/B220FITC to discriminate T-cell development from B-cell development. Fluorescence was detected by FACSCalibur flow cytometer (Becton-Dickinson). Cell marker distribution was analysed in the cellquest program. Specific fluorescence for each sample was expressed as a percentage of positively stained cells.

Analysis of mitochondrial membrane potential (MMP) with fluorescent probe

To evaluate mitochondrial depolarization, cells were incubated with a cationic dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide, Molecular Probes Inc., Eugene, OR) that exhibits potential-dependent accumulation in mitochondria (mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio, which was analysed in the cellquest program). The sensitivity of thymocytes and splenocytes to apoptosis-inducing treatment with 1 μm hydrocortisone (Hc) for 1 hr was estimated. Cells were incubated with JC-1 in complete medium (RPMI-1640 with Glutamax supplemented with 20 mm HEPES, 1 mm sodium pyruvate, 5 × 10⁻⁵ m 2-mercaptoethanol, 10% inactivated fetal calf serum).

Detection of apoptotic cells by Annexin V and propidium iodide staining

The ApoTarget™ Annexin-V FITC Apoptosis Kit (BioSource International Inc., Nivelles, Belgium) was used to detect apoptotic cells by flow cytometry. This method is based on the selective binding of Annexin-V (AnV) to the phosphatidylserine that is displayed in the outer cell of apoptotic cells and the loss of membrane integrity, which was identified by propidium iodide (PI) staining. Briefly, cells were suspended in AnV-binding buffer. AnV FITC and PI were added to the cell suspension. Cells were analysed by flow cytometry after a 15-min incubation in the dark at room temperature. The sensitivity of thymocytes and splenocytes to apoptosis-inducing treatment with 1 μm Hc for 5 hr was determined. Cells were suspended in complete medium and incubated in 24-well plates at 37° in 5% CO₂. Wells without Hc were used as controls. Cells from Ctrl and ASM-exposed mice were prepared in triplicate.

Data handling and statistics

Data are expressed as arithmetical means and SD. Analysis of variance (anova) was used to determine the significance of differences between the control and experimental groups. Probability values < 0·05 were considered significant.

Results

Reversibility of phenotype distribution changes in the thymus of mice exposed to ASM

As reported previously,^{12, 13} the percentages of single-positive (SP; CD4⁺ and CD8⁺) and TCRβ^high thymocytes were increased in mice of both sexes exposed to ASM for 72 hr. Our recent results showed the reversibility of such an effect 7 days after ASM exposure (Fig. 1). In control mice the ratio of DP thymocytes : sum of SP thymocytes was approximately 4, and the TCRβ^low : TCRβ^high thymocytes index was > 2·5. By 72 hr postexposure both indices had decreased to approximately 1·0, and 7 days postexposure they had increased to the values characteristic for the thymus of control mice.

Figure 1.

Distribution of thymocyte markers after ASM exposure. Ctrl, control mice; ASM 72 hr, 72 hr after ASM exposure; ASM 7 days, 7 days after ASM exposure. *Content of the thymocyte populations significantly differs from Ctrl ASM 7 days and from (P=0·001).

Apoptosis in central and peripheral lymphoid organs

One possible cause of such a rapid percentage increase of mature thymocytes (SP, TCRβ^high) and decrease of less mature DP thymocytes may be ASM-induced apoptotic cell death in less mature thymocyte subsets. The estimation of apoptotic cells in thymus, bone marrow and spleen did not reveal differences in the level of apoptosis measured by AnV and PI staining between control mice and those 72 hr postexposure to ASM. Changes in MMP were observed very early after the induction of apoptosis machinery and may be good markers of apoptosis pathway and kinetics. Thymocytes from mice isolated 72 hr postexposure to ASM showed an accelerated loss of mitochondrial potential, the evidence being the increase in the percentage of thymocytes in the R3 region (Fig. 2a). In vitro treatment of thymocytes with Hc revealed higher susceptibility to apoptosis induction in thymocytes from ASM-exposed mice, shown by an increase in the percentage of cells in the R3 region (Fig. 2b). The appearance of thymocytes in the R4 region after incubation with Hc points to the acceleration of the kinetics of apoptosis. Changes of MMP in splenocytes did not differ between Ctrl and ASM-exposed mice.

Figure 2.

Mitochondrial depolarization in thymocytes after ASM exposure. Ctrl, control mice; ASM, ASM-exposed mice;(a)incubation in medium alone,(b)incubation in medium with Hc. *Mitochondrial potential in ASM samples both without (a; −Hc) and with Hc (b; +Hc) significantly differs from that in the respective controls. #Mitochondrial potential in Ctrl and ASM groups with Hc significantly differs from that in Ctrl and ASM groups without Hc respectively. ^Significant difference between the Ctrl and ASM groups when comparing the results of the subtraction: (% of thymocytes with Hc) − (% of thyomcytes without Hc), respectively in R2, R3 and R4. The R2 region contains cells with normal mitochondrial potential; the R3 and R4 regions contain cells with sequential loss of mitochondrial potential, respectively, to the decrease of red/green fluorescence intensity ratio.

Induction of apoptosis in thymocytes and splenocytes from mice exposed to ASM

We estimated the induction of apoptosis in thymocytes and splenocytes after 5 hr in vitro incubation with Hc after 24, 48 and 72 hr exposure of mice to ASM to evaluate their susceptibility to this strongly apoptosis-inducing hormone (Fig. 3). The highest percentage of apoptotic thymocytes from control as well as ASM-exposed mice was found after 24 hr of exposure. The majority of thymocytes were in the early phase of apoptosis (AnV⁺ PI^– cells) independent of the presence of Hc in the medium. The percentage of apoptotic cells slowed down with the incubation time (48 and 72 hr), but was still higher for thymocytes from ASM-exposed mice incubated with Hc. No differences were found in the level of apoptosis of splenocytes from either control or ASM-exposed mice.

Figure 3.

Thymocyte apoptosis after exposure of mice to ASM. Ctrl, control mice, ASM, ASM-exposed mice; medium, AnV⁺ PI^–, 5 hr incubation in medium alone, early phase of apoptosis; medium, AnV⁺ PI⁺, 5 hr incubation in medium alone, late phase of apoptosis; Hc, AnV⁺PI^–, 5 hr incubation in medium with Hc, early phase of apoptosis; Hc, AnV⁺ PI⁺, 5 hr incubation in medium with Hc, late phase of apoptosis. *Data significantly different from Ctrl; ▾ data significantly different from samples without Hc within Ctrl and ASM groups (P=0·005).

The early phase of thymocyte development

Exposure to ASM resulted in an increase in the percentage of undifferentiated TN (CD3^– CD4^– CD8^–) thymocytes (Fig. 4) and of CD4^low thymocytes, which represent the next stage of early thymocyte development (Fig. 5). A parallel increase of c-Kit and Thy-1.2 expression in the population of thymocytes from ASM-injected mice was demonstrated (Fig. 6). In addition, a parallel increase of the expression of CD3 and CD69 was observed (Fig. 7). Finally the expression of CD132 (common γ-chain) was increased in the population of thymocytes isolated from ASM-exposed animals (Fig. 8a). The increase in the expression of CD132 was the result of an increase in the percentage of TCRβ^high thymocytes (compare histogram G4 Fig. 8c with histogram G4 Fig. 8b). Changes in the expression of CD3, CD69, Thy-1.2, c-Kit and CD132 were shown to be statistically significant according to the Kolmogorov–Smirnov statistic performed by cellquest program.

Figure 4.

Changes in the content of TN thymocytes after exposure to ASM. Ctrl, control mice; ASM, ASM-exposed mice. LL indicates lower left, position of TN thymocytes (CD3^– CD4^– CD8^–) in the quadrant, *significantly different from Ctrl.

Figure 5.

Changes in the content of CD4^low thymocytes after exposure to ASM. Ctrl, control mice; ASM, ASM-exposed mice; LL, lower left, the position of TN and CD4^low thymocytes in the quadrant; CD4^low=(TN + CD4^low) − TN; *significantly different from Ctrl.

Figure 6.

Thy-1.2 and c-Kit expression on thymocytes. Ctrl, control mice; ASM, ASM-exposed mice.

Figure 7.

CD3 and CD69 expression on thymocytes. Ctrl, control mice; ASM, ASM-exposed mice.

Figure 8.

CD132 expression on thymocytes. Ctrl, control mice; ASM, ASM-exposed mice.(a)CD132 expression in total thymocyte population (control and ASM-exposed mice combined); G1, gate for total thymocytes.(b)CD132 expression on TCRβ^– (G2), TCRβ^low (G3), and TCRβ^high (G4) thymocytes of control mice.(c)CD132 expression on TCRβ^– (G2), TCRβ^low (G3), and TCRβ^high (G4) thymocytes of ASM-exposed mice.

Changes in the distribution of bone marrow cell populations

The relative amounts of T-cell progenitors and of B cells in the bone marrow lymphoid cells were changed in mice exposed to ASM. An increase in the percentages of CD127⁺ (IL-7Rα) cells (Fig. 9) and Thy-1.2⁺ cells and a decrease in the percentage of B220⁺ cells were also observed (Fig. 10).

Figure 9.

Changes in the content of CD127⁺ cells in the bone marrow. (a) Isotypic control for non-specific fluorescence; (b) control mice, (c) ASM-exposed mice; UL (upper left), i.e. the position of CD127⁺ cells in the quadrant; *significantly different from Ctrl.

Figure 10.

Thy-1.2 and B220⁺ cells in the bone marrow. Ctrl, control mice; ASM, ASM-exposed mice; UL (upper left), the position of Thy-1.2⁺ cells; LR (lower right), the position of B220⁺ cells; *significantly different from Ctrl.

Discussion

In this paper we postulate that mice exposed to ASM under experimental conditions can evolve mechanisms responsible for the maintenance of T-cell homeostasis. When T cells are depleted, by chemotherapy, irradiation, or external agents, homeostasis-driven proliferation takes place. Constant T-cell numbers are maintained by two mechanisms: HDP of existing T cells in the periphery and de novo T-cell differentiation from stem cells in the thymus.^{18, 19} Our previous study demonstrated changes in the cortical and medullar zones in the thymus of mice exposed to high dosages of air pollutants and a lack of structural changes in the peripheral lymphoid organs.²⁰ The exposure of mice to a dose of 170 mg/kg of body weight for 72 hr demonstrated the enlargement of the medulla and simultaneous reduction of cortex (unpublished data), which may point to the vigorous process of thymocyte selection. In mice, the size of their lymphocyte populations is approximately constant and the proliferation rate and the death rate should be approximately equal. Various environmental agents exert a strong toxic effect on lymphoid cells, which results in an increase in the death rate.^{21, 22} When the death rate reaches a higher level than the proliferation rate, an additional input of cells has to be postulated to compensate for the cells that are lost. Changes in thymocyte population distribution observed after 72 hr of exposure to ASM were no longer visible by 7 days postexposure. We hypothesize that this reversibility did not result from the simple effect of ASM deprivation but was achieved by an active process that returned T cells to homeostasis. Homeostatic mechanisms are still poorly understood. They are responsible for the regulation of lymphocyte numbers at the following critical points: rate of production, alteration in life span and by division of cells.²³ The results of our studies allowed us to start with a hypothesis of accelerated thymocyte development and escape of mature cells to the periphery to compensate for the loss of cells resulting from ASM exposure. First, we demonstrated that the shift from less mature DP thymocytes to SP thymocytes was not as a result of the simple effect of apoptosis induction. The cell cycle pattern of thymocytes (as well as of splenocytes) was not changed by ASM exposure (data not presented). We did not observe differences in the level of apoptosis measured by AnV/PI staining of freshly isolated cells from thymus, bone marrow and spleen between control and ASM-exposed mice. However, we observed that mitochondrial depolarization was stronger in thymocytes from ASM-treated animals and the percentage of apoptotic cells measured by AnV/PI staining after 5 hr of incubation in medium without Hc did not differ between Ctrl and ASM-exposed mice. The apparent discrepancy between the lack of difference in the level of apoptosis of thymocytes from control and ASM-exposed mice estimated by AnV/PI staining and the decrease of MMP in thymocytes from ASM-treated animals may point to a difference in the kinetics of apoptosis resulting from ASM exposure. The collapse of MMP marks a point-of-no-return in death programming, and it represent the initial phase of apoptosis.^{24, 25} There is evidence that ultrafine particles target the mitochondria and localize there, inducing oxidative stress and damaging the mitochondria.²⁶ Several reports describe the mechanisms of apoptosis induction by heavy metals,^{27, 28} or the blockage of apoptosis by mixtures from metal-rich environments and by low metal concentrations.^{29, 30} The higher percentage of apoptotic thymocytes (AnV⁺ PI^–) from ASM mice is independent of the presence of Hc in the medium after 24 hr of incubation and may be the result of the presence of endogenous glucocorticoids induced by stress. Thymocytes from mice after different times of ASM exposure were more susceptible to the influence of Hc: 18 hr of incubation with Hc induced apoptosis in 98% of thymocytes from ASM-exposed mice compared to 80% of control thymocytes (data not presented). The increased susceptibility of thymocytes to the induction of apoptosis might be equilibrated by accelerated thymocyte development and enhanced influx of T-cell progenitors from the bone marrow. In the normal organism, apoptosis is necessary for the elimination of ‘miseducated’cells or improper/damaged cells. Deregulation of apoptosis results in the loss of homeostasis, which in turn must be maintained for an adequate balance between different cell types.^{31, 32} T-cell homeostasis in young mice is achieved by continuous exportation of newly formed mature T cells from the thymus.^{33, 34} The earliest thymic progenitors, CD4^low CD8^– CD44⁺ CD25^– c-Kit⁺ Thy-1^low, become, after TCRβ and TCRα rearrangement, TCR^low CD4⁺ CD8⁺.^35–37 At this stage, they are ready to undergo positive selection. These cells migrate to the thymus, and the majority of them are T-cell committed. Analysis of thymocyte subset content revealed an increase in the percentage of TN (CD3^– CD4^– CD8^–) and CD4^low cells in ASM-exposed mice. The simultaneous increased expression of Thy-1.2 and c-Kit on thymocytes from experimental animals points to the process of thymocyte maturation (a shift from Thy-1.2^low to Thy-1.2^high), and the influx of T-cell progenitors characterized by the expression of c-Kit. The maturational transition from CD4⁺ CD8⁺ to CD4⁺ CD8^– and CD4^– CD8⁺ cells is restricted to TCRβ^high and CD3^high thymocytes and the increase in CD69 expression is related to the positive selection process.^{16, 17} The parallel increase in the expression of CD3 and CD69 was more pronounced in ASM-exposed mice. In addition, the increase in the expression of CD132 on thymocytes from mice exposed to ASM also points to an acceleration of T-cell development. CD132 is the common component of the receptor complex for IL-2, IL-4, IL-7, IL-9, IL-15. IL-7 is thought to be the crucial cytokine responsible for peripheral homeostatic expansion and for the regulation of thymopoiesis.^{38, 39} The lack of IL-7 or CD127 blocks the development of T and B cells. The thymus should be continuously supplemented by T-cell progenitors from the bone marrow. Analysis of the early lymphoid progenitors in the bone marrow of ASM-exposed mice showed an increase in the percentage of cells expressing the CD127 characteristic for the common lymphoid progenitors. Among these cells the percentage of Thy-1.2⁺ cells was increased. Our results suggest that the development pathway of T-cell progenitors is favoured in ASM-exposed mice.

Conclusions

In mice exposed to ASM we observed the following:

1. the reversibility of the changes in distribution of thymocyte populations, which was not the result of a simple effect of ASM deprivation, but was an active process, returning T-cell homeostasis;

2. the same percentage of apoptotic cells in lymphoid organs as in control mice;

3. the increased loss of MMP in thymocytes, which may point to a difference in the kinetics of apoptosis induction;

4. the increase in susceptibility to Hc-induced apoptosis in thymocytes;

5. the enhancement of the positive selection process and thymocyte development (increased expression of Thy-1.2, CD3, CD132 and CD69);

6. symptoms of accelerated influx of immature T cells to the thymus marked by the increase in c-Kit expression, the percentage of TN thymocytes (CD3^– CD4^– CD8^–) and CD4^low thymocytes;

7. the increase of the percentage of CD127-positive cells in the bone marrow, which confirms the enhancement of lymphoid cell development; the increase in Thy-1.2-positive cells in the lymphoid bone marrow population suggests that the development pathway of T-cell progenitors was favoured in ASM-exposed mice.

Taken together, these results put a new light on the effect of xenobiotics on T-cell homeostasis and confirm our hypothesis that there is acceleration of thymocyte development under conditions of elevated risk resulting from contact with environmental toxic agents. This process compensates for the loss of cells resulting from exposure to ASM and contributes to the maintenance of T-cell homeostasis.

Abbreviations

ASM

airborne suspended matter

DP

double positive

Hc

hydrocortisone

HDP

homeostasis-driven proliferation

MMP

mitochondrial membrane potential

SP

single positive

TN

triple negative