Abstract
In 2005, the International conference on harmonization (ICH) recommended that all new human pharmaceuticals be tested for unintended immunomodulatory potential via a tiered approach. Included in this approach is a semiquantitative description of changes in the separate compartments of lymphoid tissue (also called enhanced histopathology). Chlorambucil was administered to Hanover Wistar rats at regular time points, followed by a treatment-free (recovery) period. Groups of treated and control animals were sacrificed regularly during both the treatment and recovery periods. Selected tissues were removed, weighed fresh and fixed in formalin, processed, and stained with hematoxylin and eosin. Blood samples and bone marrow smears were also obtained. With the use of enhanced histopathology, a description of the changes in lymphoid tissues and bone marrow was used as a means of assessing the susceptibility, and recovery, of the different lymphoid cell populations over time. A correlation with organ weights, flow cytometry data, and bone marrow cytology was achieved. The administration of chlorambucil in the Hanover Wistar rat provided a useful tool to examine the rate and sequence of changes in the lymphoid organs and bone marrow during treatment with, and the recovery from the effects of, a potent immunosuppressive agent.
Introduction
Immunotoxicity is the direct or indirect adverse effect of a chemical on the immune system resulting in suppression, or enhancement, of the immune response. Suppressing the immune response can lead to decreased host resistance to infectious agents or tumor cells, whereas enhancing it can result in autoimmune diseases or hypersensitivity (Descotes 2006). Immunotoxicity may be accompanied by changes in the weight of lymphoid organs and/or alterations in the normal microscopic architecture. In 2005, the International Conference for Harmonization (ICH) produced a guideline on immunotoxicity studies for human pharmaceuticals (ICH S8), which recommends that all new human pharmaceuticals be tested for immunotoxic potential. The ICH S8 document is restricted to unintended immunosuppression or immunoenhancement of human pharmaceuticals and avoids drugs, such as anticancer agents, where effects on the immune system are a predicted result of secondary toxicity to the bone marrow and lymphoid organs. This document has focused attention on an already growing interest in investigative toxicology methods for the evaluation of drug- and chemical-induced effects on the immune system (Germolec et al. 2004; Haley 2003; Haley et al. 2005; Kuper et al. 2000). Kuper and coworkers, in particular, have published suggested approaches to histological evaluation of the anatomically and functionally distinct cell subpopulations within each of the lymphoid tissues (also called enhanced histopathology)(Kuper, Schuurman, and Vos 1995; Kuper et al. 2000; Kuper et al. 2002). In 2006, a special issue of Toxicologic Pathology concentrated on the histomorphology of lymphoid tissues and bone marrow and the enhanced histopathology of the immune system (Maronpot 2006).
The objective of the present study was to carry out microscopic examination of the lymphoid organs and bone marrow of the rat following repeat dose exposure to a known immunosuppressive agent. It was of particular interest to investigate the use of enhanced histopathology of the lymphoid tissues both during the treatment and posttreatment (recovery) periods as a means of assessing the susceptibility and recovery of the different lymphoid cell populations over time.
The anticancer drug chlorambucil (CHB), marketed in the United Kingdom as Leukeran ( British National Formulary 2007; Medicines Compendium 2002), is a cytotoxic compound that is used widely in cancer chemotherapy (Dollery 1999; Sweetman 2002). The major indication for the therapeutic use of CHB is the treatment of chronic lymphocytic leukemia (Parfitt 1999), where the effect of CHB is to lower the proportion of lymphocytes in the bone marrow, reduce the peripheral blood lymphocyte count, and reduce the size of the spleen and lymph nodes (Dollery 1999).
Since the hematotoxic effects induced by CHB in the female Hanover Wistar rat had previously been examined (Molyneux et al. 2004) and an effective regimen of repeated drug administration at a maximum tolerated dose had been identified, it was decided to use this compound in the present investigation as a model immunotoxicant. The aims of the present study were, therefore, to use a known immunotoxicant dose regimen of CHB to study the differential toxicity to the lymphoid organs during the atrophic and subsequent recovery phases of drug administration, using the principles of enhanced histopathology to differentiate sensitive from resistant cell populations.
Materials and Methods
Animals
Female Hanover Wistar rats (B and K Universal Ltd, Grimston, Aldbrough, Hull, UK) were caged in groups of three to five, bedded on wood shavings, and given diet (Rat and Mouse No. 1; SDS Ltd, Witham, Essex, UK) and water ad libitum. The temperature was maintained at 19 °C to 21 °C with a relative humidity of 45% to 65%; lighting was on a twelve-hour light: dark cycle (lights on at 7:00 a.m.). Animals were observed at least once daily for clinical evidence of ill health and signs of drug toxicity, and they were weighed at intervals of no longer than seventy-two hours apart (Table 1). Animal procedures were carried out under local ethical committee guidelines and approval, and followed the U.K. Home Office (1989) “Code of Practice for the Housing and Care of Animals used in Scientific Procedures.”
Chlorambucil Administration
Chlorambucil (Sigma Chemical Co., Poole, Dorset, UK), was dissolved in 10.0 mL acetone at a concentration of 4.35mg/m:. Immediately before administration, 20 mL of deionized water was added to 10 mL of the CHB–acetone solution, which was administered by intraperitoneal (IP) injection at 6.3 mg/kg (in a dose volume of approximately 0.5 mL/rat); control animals were treated with the acetone/water solution (vehicle) at the same dose volume and by the same route. Animals received single IP injections on two, four, or six occasions over a treatment period of seventeen days.
Experimental Design
The experimental design is summarized in Table 1. Fifty-five rats, with a mean body weight 155.4 g, were divided into two groups of thirty-one CHB-treated rats (mean body weight 158.6 g) and twenty-four vehicle-treated controls (mean body weight 151.2 g).
On six occasions over the seventeen-day treatment period, animals received 6.3 mg/kg CHB or vehicle (on days 1, 3, 8, 11, 14, and 17). On days 4 (after two doses) and 12 (after four doses) of the treatment period, and on days 1, 5, 9, and 15 after the final (sixth) CHB dose (i.e., in the subsequent recovery period, when animals received no treatment), animals from the vehicle-treated control group (n = 4) and the CHB-treated group (n = 5 or 6) were killed, and tissues, bone marrow, and blood were examined.
Autopsy Procedures and Sample Collection
Rats were killed by IP injection of pentobarbitone sodium (Sagatal; Rhône Mérieux Ltd, Harlow, Essex, UK) and exsanguinated from the abdominal aorta. A 1.0-mL aliquot of blood was anticoagulated with 1.5 mg/mL dipotassium EDTA (Teklab, Sacriston, Durham, UK). A femoral bone marrow suspension was prepared; the end of the left femur was removed at the epiphysis, and the marrow was flushed into 5.0 mL of Iscove’s modified Dulbecco’s medium (IMDM) (Life Technologies, Paisley, UK) supplemented with 10.0% fetal calf serum (PAA Laboratories GmbH, Linz, Austria) to give a marrow cell suspension, which was placed on ice. A marrow smear was prepared from the contents of the left tibia, as previously described (Turton et al. 2006). After drying in air at room temperature, smears were fixed in 100% methyl alcohol for one hour and air-dried prior to staining. The spleen and thymus were removed, weighed, and placed in 10.5% 0.1 M phosphate buffered formalin fixative for a minimum of two weeks. Other tissues (sternum; right femur; small and large intestines, with surrounding mesentery and mesenteric lymph nodes; and mandibular lymph nodes), were removed and placed in fixative for a minimum of two weeks.
Hematology and Bone Marrow Smears
The peripheral blood EDTA samples, taken on days 1 and 15 of the recovery period, were analyzed with the Advia 120 hematology analyzer with rat-specific software (Bayer/Siemens, Newbury, Berks, UK) to generate a complete blood count. Peripheral blood EDTA samples were also stained for lymphocyte subsets (B, T, CD4, and NK cells). Femoral bone marrow suspension from rats on days 1 and 15 of the recovery period was maintained in IMDM prior to flow cytometry analysis with the FACS Calibur (Becton Dickinson Biosciences, Oxford, UK). Flow cytometry acquisition was carried out within twelve hours of sampling according to the method of Saad et al. (2001) for bone marrow myeloid, erythroid, and lymphoid precursor percentages, and myeloid:erythroid (M:E) ratios.
For bone marrow staining, 100 μL of bone marrow suspension was adjusted to 1 × 107/mL cells and stained with 5 μL CD45 fluorescein (FITC) and 10 μL CD71 phycoerythrin (PE) (AbD Serotec UK Ltd, Oxford, UK) for twenty minutes at 4°C. After 1 mL of PBS containing 0.5% bovine serum albumin (BSA) (v/v) was added, the sample was mixed and centrifuged at 300 × g for five minutes. Following removal of the supernatant, the resulting cell pellet was resuspended in 400 μL PBS/BSA with 3.3 × 107g/mL of the DNA stain laser dye styryl-751 (LDS751) (Molecular Probes, Eugene, OR, USA) and incubated at room temperature for at least twenty minutes before evaluation on the flow cytometer (see below).
For peripheral blood staining of lymphocyte subsets, 100 μL of whole blood was incubated with 0.5 μg of each of the following: anti-rat CD45RA:PE (B cells), CD4: allophycocyanin (APC) (helper T cells), TcR peridinin-chlorophyll-protein complex (PerCP) (T cells), and NKRP1A FITC (Pharmingen, Oxford, UK) (NK cells) for twenty minutes at 4°C. After 1 mL of PBS/BSA (v/v) was added, the sample was centrifuged at 300 × g for five minutes. The cell pellet was resuspended in 2 mL FACSLyse (Becton Dickinson Biosciences) and left for twenty minutes at room temperature. The sample was centrifuged at 300 × g for five minutes before being resuspended in 400 μL PBS/BSA before evaluation on the flow cytometer.
Samples were evaluated on a BD FACSCalibur flow cytometer (Becton Dickinson Biosciences) equipped with an argon ion laser at 488 nm excitation, a red diode laser at 635 nm excitation, and the appropriate filters for excitation and emission of the fluorochromes and stains used (Becton Dickinson, San Diego, CA, USA). Data analysis was done after acquisition using CELLQuest software (Becton Dickinson Biosciences).
Bone marrow samples were acquired using the gating strategy of Saad et al. (2001) and data saved for 10,000 nucleated cells. Data generated were expressed as percentage of nucleated cells acquired.
Peripheral blood lymphocyte subset data were acquired gating the total lymphocyte population based on scatter properties and data saved for 3,000 lymphocytes. Data generated were expressed as percentage of lymphocytes. All absolute lymphocyte count data were generated using the Advia 120 hematology analyzer.
Microscopic Examination
The tissues selected for examination were sections of bone marrow from the right femur and the sternum, spleen, thymus, mandibular and mesenteric lymph nodes, and Peyer’s patches within the ileum. Following fixation, the right femur and sternum were decalcified in 10% formic acid for approximately five days, after which they were washed thoroughly in running tap water and processed along the same schedule as the other tissues. All tissues were trimmed to blocks no thicker than 3 mm, dehydrated in graded industrial methylated spirit, cleared in xylene, and processed to paraffin wax on a Shandon Excelsior Tissue Processor (Thermo Fisher Scientific, Runcorn, UK) using our standard rodent tissue schedule prior to sectioning at 4 to 5 μm. Sections were dewaxed and brought through a graded series of ethanols to distilled water, stained with hematoxylin and eosin (H&E), and examined by light microscopy.
Examination of H&E-stained bone marrow sections simply involved the assessment of levels of cellularity. No interpretation of the separate cell components of the bone marrow was carried out on the tissue sections because of the parallel flow cytometric data and the smears. Chlorambucil-related changes in the spleen, thymus, lymph nodes, and Peyer’s patches were graded with respect to controls. Increases in cell populations over the respective control group (0) were given a positive score, based on severity. Decreases in cell population over the respective control group (0) were given a negative score, based on severity. Severity scores were given as numerical grades of 1 to 4 (minimal, mild, moderate, and marked, respectively). Values were entered into an Excel spreadsheet, and graphs representing changes over time were created from these data.
Tibial bone marrow smears were prepared on days 4 and 12 of the treatment period and on days 1, 5, 9, and 15 of the recovery period. Smears were stained with May Grünwald Giemsa and examined by light microscopy. Smears from CHB-treated animals were scored relative to vehicle controls for qualitative morphological changes (apoptosis and the maturation and differentiation of erythroid and granulocytic series, and the megakaryocytic populations).
Immunohistochemistry
Selected sections of thymus were processed for the immunohistochemical expression of cleaved caspase 3 as a marker of apoptosis. Paraffin sections (4–5 μm) of thymus were cut and mounted on SuperFrost Plus electrostatically charged glass slides (Thermo Fisher Scientific, Runcorn, UK). Sections were allowed to dry at 37°C overnight in an incubator and were dewaxed in xylene, passed through graded alcohols, and rehydrated. Heat-mediated antigen retrieval was performed in a Milestone RHS-2 microwave (Milestone, Sorisole, Italy) at 110°C for two minutes in 1 mM EDTA buffer, pH 8.0.
Immunohistochemical staining was performed, at room temperature, on a Lab Vision Autostainer 720 (Lab Vision, Newmarket, UK). The antibody diluent and wash buffer was 0.05 M Tris-buffered saline plus 0.05% Tween 20 (TBST), pH 7.6. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in TBST for ten minutes. Slides were washed and incubated for twenty minutes with 5% normal goat serum (Dako UK Ltd, Ely, UK) in TBST. Excess blocking serum was blown off, and the slides incubated for sixty minutes with polyclonal rabbit anti-human cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA.) diluted 1:50 in TBST. Following washing, the slides were incubated for thirty minutes in rabbit-specific EnVision+ System–HRP (Dako UK Ltd), and visualized by incubation in diaminobenzidine (DAB), from the EnVision+ kit, for ten minutes. The slides were counterstained for one minute using Carazzi’s hematoxylin (Clin-Tech, Guildford, UK). Negative control slides were simultaneously processed by substituting a rabbit immunoglobulin (Ig) fraction, diluted to the same Ig concentration, for the primary antibody, or by omission of the primary antibody. Stained sections were dehydrated and mounted under glass coverslips with Histomount (R.A. Lamb, Eastbourne, UK).
The demonstration of extramedullary hematopoiesis (EMH) in spleen sections was achieved by immunohistochemically staining for myeloperoxidase using the methodology already outlined, but with polyclonal rabbit antihuman myeloperoxidase (Dako UK Ltd), diluted 1:500 in TBST, as the primary antibody.
Statistical Analysis
Body weight and organ weight data from CHB-treated and vehicle-treated control groups were compared using the Student t test for unpaired samples (Microsoft Excel; version 9.0, 1999, Microsoft Ltd, Microsoft UK, Reading, UK).
Results
Clinical Signs and Body Weight Changes
Mean group body weights of vehicle control and treated animals are presented in Table 2. Control animals increased in mean body weight from 158.8 g on day 4 of the treatment period to 160.0 g on day 12 of the treatment period, an increase of 0.8%.
For CHB-treated animals, the mean body weight on day 4 of the treatment period was 150.0 g, and on day 12 of the treatment period it was 156.8 g, an increase of 4.5%. From day 1 to day 15 of the recovery period, control mean body weight increased from 179.0 g to 207.0 g (15.6%). The mean group body weights of CHB-treated animals were lower at all time points, both during treatment (6.0%) and recovery (18.1%), when compared with concurrent vehicle controls. Reductions were statistically significant on day 4 (p < .05) of the treatment period and days 1 (p < .01) and 5 (p < .05) of the recovery period.
Gross Findings at Autopsy and Relative Spleen and Thymus Weight Changes
Slight thickening of the mesentery, with or without fibrin deposits and adhesions, occurred in a proportion of control and treated animals from all groups. These changes were also seen in animals from the recovery period. Macroscopically, the spleen and thymus were reduced in size in CHB-treated animals on day 12 of the treatment period, but not on day 4, and days 1 and 5 of the recovery period (groups 2, 3, and 4).
The group mean relative spleen and thymus weights from vehicle-control and treated animals are presented in Table 2. Group mean relative spleen weights from CHB-treated animals on days 4 and 12 of the treatment period (groups 1 and 2) were significantly reduced, namely, 65.6% (p < .001) and 40.7% (p < .001) of the concurrent vehicle-control weights, respectively. Spleen weights from CHB-treated animals progressively returned to normal over the recovery period. On recovery days 1, 5, and 9 (groups 3, 4, and 5), weights were 62.1% (p < .001), 84.8% (p < .05), and 98.7% (NS) of concurrent controls, respectively.
The group mean relative thymus weights from CHB-treated animals on days 4 and 12 of the treatment period (groups 1 and 2) were significantly reduced to 65.6% (p < .001) and 37.2% (p < .001) of the concurrent control values, respectively. These decreased values for relative thymic weight in CHB-treated animals continued during the recovery period. On day 1 and day 5 of the recovery period (groups 3 and 4), mean thymus weights were 37.7% (p < .001) and 36.6% (p < .001) of the mean control values, respectively. There was a return toward normal values by days 9 (group 5) and 15 (group 6) of the recovery period, when weights were 74.2% (NS) and 95.3% (NS) of the mean control weights, respectively.
Bone Marrow Cytology and Flow Cytometry
Examination of bone marrow smears from CHB-treated animals revealed an initial increase in nuclear anomalies in late-stage erythroid precursors on day 4 of the treatment period (group 1), including increased nuclear fragmentation and binucleation (Figure 1a and 1b; Table 3). Increased levels of apoptosis were also apparent. These changes were most pronounced on day 12 (group 2). There were no morphological changes in the granulocytic series on day 4 of the treatment period (group 1), but a mild increase in immature erythroid and granulocytic precursors was noted on day 12 of treatment (group 2) (Figure 1c).
On day 1 of the recovery period (group 3), bone marrow smears were characterized by an increase in immature granulocytes and an increased M:E ratio. Mild nuclear anomalies in late-stage erythroid precursors persisted until day 5 of recovery (group 4) but was not seen after this time (Table 3). There were mild increases in early erythroid and granulocytic precursors, but no increased apoptosis of the erythrocyte lineage in bone marrow smears of CHB-treated animals on day 9 of the recovery period (group 5). At the end of the fifteen-day recovery period (group 6), the appearance of the bone marrow smears from CHB-treated animals was normal. These changes were confirmed by flow cytometry (Table 4).
Flow cytometry findings on day 1 of the recovery period (group 3) demonstrated that lymphocytic changes were characterized by decreased total numbers in the bone marrow and a reduced peripheral blood total lymphocyte count. Flow cytometry data on peripheral blood lymphocyte subsets in these animals showed that this finding was associated with a decrease in the number of B and T cells, but that the number of NK cells was unaffected.
At the end of the fifteen-day recovery period (group 6), the decreased numbers of lymphocytes in the bone marrow (Figure 2) and in peripheral blood total lymphocyte counts persisted. In the peripheral blood, this decrease was a result of reductions in the T lymphocyte counts.
Histopathology
Bone Marrow:
The changes in cell populations over time are represented in Figure 3. Microscopic changes in sections of bone marrow from the sternum and femur were comparable. There was an immediate decrease in the hematopoietic cell population in all CHB-treated animals at the earliest time point sampled, day 4, which persisted at day 12 of the treatment period (groups 1 and 2), when compared with respective controls (Figure 4a, 4b, and 4c). Megakaryocytes were the most resistant cell and the last cell type within the bone marrow to be affected. A small number of these cells showed degeneration in animals on day 4 of the treatment period (group 1), and they were severely decreased in number on day 12 (group 2). During the recovery period, a progressive return to normal levels of bone marrow cellularity occurred between days 1 (group 3) and 5 (group 4). (Figure 4d and 4e), with all cells showing similar degrees of recovery.
An increase in adipocytes, concurrent with the decrease in hematopoiesis, was also evident from day 12 of the treatment period (group 2). Fat persisted in the marrow of a proportion of animals whose hematopoietic cell population had returned to within normal limits on day 5 of the recovery period (group 4). The presence of vacuoles (presumptive fat) was also noted in marrow smears from a single group 3 animal on day 1 of recovery (Figure 4d).
Thymus:
Thymic changes, expressed over time, are presented in Figure 5. On day 4 of the treatment period (group 1), the thymus showed a reduction in the corticomedullary ratio, with prominent apoptosis of the cells of the cortex and, to a lesser extent, the medulla (Figure 6a and 6b). Apoptosis of lymphoid cells was confirmed immunohistochemically by cleaved caspase 3 staining (Figure 7a). Apoptosis was no longer evident on day 12 in either compartment of the thymus, but there was a greater depletion of lymphoid cells, particularly in the cortex, with loss of the normal corticomedullary demarcation (Figure 6c and 6d).
On day 1 of the recovery period (group 3 animals), the lymphoid population of the thymus remained noticeably depleted and the corticomedullary demarcation was indistinct. Tingible body macrophages (containing intracytoplasmic apoptotic lymphocytes) occurred within the cortex, and to a lesser extent the medulla, in some animals. However, at the first time point following recovery day 1 (group 3), there was already evidence of partial recovery characterized by the presence of large lymphoblastic cells and of mitotic figures in both areas (Figure 6f). On day 5 of the recovery period (group 4), cellular depletion was still present, if less evident, and there was a greater increase in lymphoblasts within both the cortex and the medulla (Figure 8a and 8b). In addition, mitotic figures were frequent. Tingible body macrophages were no longer a major feature, but they remained present on day 9 of the recovery period (group 5). The most striking low-magnification feature of the thymus at this time point, however, was an increase in the corticomedullary ratio (Figure 8c and 8d). A return to a normal thymic morphology was seen on day 15 of the recovery period (group 6) (Figure 8e and 8f).
Spleen:
Splenic changes over time are presented in Figure 9. In the white pulp of CHB-treated rats (Figure 10a), prominent apoptosis occurred within the lymphoid follicles, the marginal zone lymphocytes, and, to a lesser extent, the periarteriolar lymphoid sheath (PALS) on day 4 of the treatment period (group 1). There was a corresponding progressive decrease in white pulp cellularity in treated animals on days 4 and 12 of the treatment period (groups 1 and 2).
Lymphoblastic cells were seen within the PALS on day 1 of the recovery period (group 4), and full recovery of the white pulp cellularity was seen in some, but not all, animals at the end of the fifteen-day recovery period (group 6).
A similar pattern of change occurred in the red pulp of CHB-treated rats, in which the degree of EMH was reduced in all animals on days 4 and 12 of the treatment period (groups 1 and 2) when compared with respective controls. However, partial recovery of EMH was seen on day 1 of the recovery (group 3). The level of EMH returned to within normal limits, but with a predominance of myelopoiesis, on days 5 (group 4) and 9 (group 5) of the recovery period (Figure 10d). On day 15 of the recovery period (group 6), there was a notable individual variation in the level of EMH, which, in some animals, was greater than that seen in the corresponding controls.
Inflammation of the splenic capsule was an incidental finding in the spleen, both in occasional CHB-treated and control animals. This inflammation was a probable extension of the peritonitis caused as a result of the IP administration route used in the experimental protocol, corresponding with the observed thickened mesentery.
Lymph Nodes:
In CHB-treated animals, the nature of the changes seen in the mesenteric and mandibular lymph nodes was similar during the treatment period (results not shown). Changes on day 4 of the treatment period (group 1) were characterized by an early loss of germinal centers, as well as increased apoptosis within the cortex and to a lesser extent the paracortex. By day 12 of the treatment period (group 2), the severity of lymphoid depletion within the cortex and paracortex was increased. In addition, at this time point, there was a severe reduction in the cellularity within the medullary sinuses, and plasma cells were the predominant lymphoid cell at this site. However, subtle differences were seen between the mesenteric and mandibular lymph nodes during the recovery period. On day 1 of the recovery period (group 3), cellular depletion was severe in the mandibular nodes, with only stromal cells and a few plasma cells remaining, whereas in the mesenteric nodes, small numbers of lymphoblasts were also present. On day 9 of the recovery period (group 5), changes in nodes from both locations were characterized by diffuse and focal cortical and paracortical infiltrates of lymphoblastic cells and a prominent increase in mitotic figures. In the mandibular node, there was also a marked increase in the number of plasma cells within the medullary sinuses. Little change was seen in the histological appearance of the mandibular nodes from CHB-treated animals on days 9 and 15 of the recovery period (group 5 and 6).
By day 15 of the recovery period (group 6), the level of cellularity within the mesenteric node had approached normal limits and included a significant population of macrophages, characteristic of lymph nodes from this site.
Discussion
Administration of CHB to Hanover Wistar rats, at the dose level used in the current study, resulted in a rapid decrease in peripheral blood lymphocytes and a profound cellular depletion of the bone marrow, the mesenteric and mandibular lymph nodes, the spleen, and the thymus. Cellular depletion was accompanied by significant decreases in the organs weighed at necropsy (spleen and thymus).
Treatment-related effects were evident in all tissues examined at the earliest time point, and in all cases, the earliest signs of toxicity were associated with prominent apoptosis of the affected cells. This finding is consistent with the mode of action of CHB, which is a bifunctional alkylating agent, resulting in the induction of DNA strand cross-linking, the consequent inability of DNA to separate during cell division, leading to the death of the cell (Hegab and Al-Mutawa 2000). Apoptosis was accompanied by depletion in cellularity. In the case of the thymus, a profound, transient rebound effect, with an increase in the corticomedullary ratio, was observed.
Histologically, recovery was seen first in the bone marrow on day 5 of the recovery period. Similar changes have been reported in man, where bone marrow suppression is the main toxic effect of CHB, and which, with normal therapeutic use, is readily, and rapidly, reversible (Gauthier and Trachtman 1999; Molyneux et al. 2004). Next to recover were the thymus and mandibular lymph nodes (recovery day 9), followed by the mesenteric lymph node (recovery day 15). In the majority of CHB-treated animals, splenic architecture did not recover to within normal limits by the end of the fifteen-day recovery period.
In CHB-treated animals, the bone marrow cellularity was significantly depleted on day 12 of the treatment period (group 2), and smears showed that changes in the erythroid series were the earliest to occur and the last to recover. Megakaryocytes were the most resistant cell type in the bone marrow, and the last to be affected, but unlike other reports of megakaryocyte resistance to CHB (Elson, Galton, and Till 1958), proportions of these cells showed degeneration on day 4 of the treatment period and were severely decreased by day 12 of the treatment period.
Histological evidence of the onset of recovery in the bone marrow was seen at day 1 of the recovery period, and the bone marrow returned to within normal limits in the majority of animals on day 5. Bone marrow smear and hematology data characterized the onset of bone marrow recovery as an increase in granulopoiesis. There was a progressive increase in fat, seen initially on day 4 of the treatment period, reaching a peak on day 12 of the treatment period and subsequently diminishing during the recovery period. This finding has been previously reported in studies with CHB (Molyneux et al. 2004). The bone marrow hematopoietic environment contains a variety of cell types, of which adipocytes are a normal component. Marrow adipocytes act as a mechanical buffer that occupies or releases space within the marrow cavity in response to changing demands for hematopoiesis. The extent of marrow fat varies inversely with the degree of hematopoiesis and the rate of cell delivery to the bloodstream. Both the local marrow microenvironment and systemic humoral factors influence hematopoieses to stimulate or suppress the proliferation and differentiation of cell lineages (Gasper 2000). Bone marrow adipose tissue has been shown to exhibit considerable plasticity and to secrete a broad spectrum of hormones, growth factors, and cytokines that can influence hematopoiesis, immune function, inflammation, and bone remodeling (Laharrague and Casteilla 2007). Under circumstances that are not clearly understood, bone marrow suppression may lead to irreversible bone marrow failure (Rudd, Fries, and Epstein 1975). However, in the present study, the replacement of marrow with adipose tissue appeared to be a less severe effect than that often seen with other myelotoxic agents, such as cyclophosphamide, busulphan, and thioacetamide, in which there is severe disruption of the stroma, with hemorrhage and possible myelofibrosis (Nath and Shaw 2007).
The thymus was the first of the lymphoid tissues in CHB-treated animals to recover, and repopulation by large immunoblasts was seen as early as day 1 of the recovery period. Microscopic indication of recovery was seen ahead of recovery of the organ weight, which progressively increased from day 9 of the posttreatment period, although remaining significantly depressed at the termination of the experiment. In the majority of animals, thymic microscopic architecture was restored to within normal limits by day 9 of the recovery period.
In the spleen, microscopic evidence of repopulation of the white pulp was not seen until day 9 of the recovery period, in the follicles (B cell–rich areas) and to a lesser extent the PALS (T cell–rich areas). Regeneration of the immune system relies on the local bone marrow environment as the supply of most of the hematopoietic stem cells (HSC), and on systemic humoral factors to promote proliferation (Meyer and Harvey 1998). Both B and T cells arise from bone marrow precursor cells. However, the generation of mature T lymphocytes also relies on the thymus, to which HSC-derived progenitors migrate and undergo T cell development and T cell receptor selection (thymopoiesis). It is therefore logical that recovery of lymphoid populations in the spleen should be seen later than in the thymus. Extramedullary hematopoiesis in the red pulp was reduced, but not completely prevented, by treatment during the CHB-treatment period, and again, the first indications of recovery occurred on day 1 of the recovery period. In addition, myelopoiesis was prominent in the recovering spleen and bone marrow. These changes are consistent with reports that at therapeutic dose levels in man, CHB induces severe lymphocytopenia, but it has a lesser effect on neutrophils, red blood cells, and platelets (Reynolds 1993).
In the lymph nodes examined, morphological changes in CHB-treated animals suggested a greater susceptibility of B cells than T cells to the toxic effects of the drug. In addition, the degree of differentiation of the B cells was important, since there was a relative sparing of the plasma cell populations. Furthermore, these lymph node findings are consistent with the report by Hegab and Al-Mutawa (2000), which stated that in addition to producing profound effects on human lymphocyte populations, high doses of the drug also targeted B cells before having any effect on T-cell populations. However, long-term administration of lower doses of the drug was found to have an equal, or greater, effect on T-cell function.
It is considered that the results of the present study in the rat will help to illustrate the practical value of the semiquantitative description of changes within the anatomical and functional compartments of the lymphoid system (also called enhanced histopathology) as a first step toward understanding the dynamic changes occurring within the immune system on exposure to, and subsequent recovery from, immunotoxicants.