Apoptotic Thymocytes Research Articles

FRACTALKINE INCREASES APOPTOTIC CELL CLEARANCE BY UPREGULATING MFG-E8 IN MACROPHAGES

Clearance of apoptotic cells is crucial in sepsis. We have previously shown that administration of immature dendritic cellderived exosomes containing milk fat globule epidermal growth factor-factor VIII (MFG-E8) to septic animals promoted phagocytosis of apoptotic cells and improved survival. MFG-E8 is an opsonin for apoptotic cells and acts as a bridging protein between phosphatidylserine on apoptotic cells and integrins on phagocytes. Fractalkine (Fr) is a CX3C-chemokine (both a chemoatractant and an adhesion molecule) that is upregulated by proinflammatory cytokines and has been proposed to play a role under inflammatory conditions. A recent study has shown that Fr increased MFG-E8 gene transcription in microglia. We hypothezised that Fr upregulates MFG-E8 in peritoneal macrophages and promotes apoptotic cell clearance without inducing inflammation. To study this, rat peritoneal macrophages (pMφ) were isolated and cultured. MFG-E8 expression was assessed by Western blot after incubation with either LPS or Fr (without LPS). The ability of macrophages to engulf apoptotic thymocytes (A0TC) was determined by TUNEL. Cytokine secretion was assessed by ELISA in cultured cell supernatant. Our results have shown that recombinant MFG-E8 (1 μg/ml) doubles phagocytosis of A0TC by pMφ. Further results are shown in the table below (PI = phagocytosis index): We found similar results in the murine RAW 264.7 cell line, indicating that fractalkine is an important prophagocytic factor. We conclude that fractalkine enhances clearance of apoptotic cells by induction of MFG-E8 in macrophages. Since it does not induce a proinflammatory response in vitro by itself, fractalkine may be safe for the use in animal models of sepsis (NIH R01 GM057468).Table

Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis

Many scientists are focusing on the apoptotic-necrotic or the necrotic-apoptotic switch and its mechanism, but little attention has been paid to the viable-apoptotic switch. Most of the techniques and methods used for detecting apoptosis are performed on fixed samples, yielding static information of specific time points. We have studied the viable-apoptotic switch in S-nitrosoglutathione (GSNO)-induced mouse thymocyte apoptosis in real-time by means of a novel technique, intensified charge coupled device (ICCD)-based real-time fluorescence micro-imaging, coupled with Annexin V-FITC labeling for phosphatidylserine (PS) translocation in cell membrane. We have successfully recorded the initiating time points (mostly at 2 h) of the viable-apoptotic switch in GSNO-initiated apoptosis, as well as shown the real-time differences between living and apoptotic thymocytes. These findings suggest that NO is also a switch molecule for the conversion from viable to apoptotic cell. Thymocytes cotreated by N-G-monomethyl-L-arginine acetate salt (L-NMMA) provide further evidence for this suggestion, as well as for the suggestion that L-NMMA prolongs the early stage of thymocyte apoptosis rather than strongly blocks it.

Phagocytosis of apoptotic and necrotic thymocytes is inhibited by PAF-receptor antagonists and affects LPS-induced COX-2 expression in murine macrophages

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.

Phospholipase D as a potential target for the antiproliferative effects of polyunsaturated fatty acids in rat thymocytes

The effects of various saturated and unsaturated fatty acids (FAs) on the proliferative response and phospholipase D (PLD) activity of rat thymocytes were investigated. When added to culture medium as complexes with albumin, all the FAs tested, except stearic acid, inhibited the ConA-induced thymocyte proliferation, eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids being the most inhibitory. Apart from 22:6n-3 which slightly increased the percentage of late apoptotic and necrotic thymocytes in the presence of mitogen, none of the FAs induced significant apoptosis or necrosis. A short 2-h preincubation of rat thymocytes in the presence of FA–albumin complexes was sufficient to induce a significant enrichment of cell phospholipids with each FA and to stimulate thymocyte PLD activity. However, 20:5n-3 was inactive despite a large enrichment in phospholipids. Furthermore, the PLD activity of activated thymocytes was negatively correlated to the proliferative response, with the exception of 20:5n-3-supplemented cells. These results support further our current hypothesis that PLD activity conveys antiproliferative signals in lymphoid cells, and suggest that 20:5n-3 inhibits thymocyte proliferation by a particular mechanism unrelated to that of the other FAs.

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane

Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.

Regulation of T‐cell progenitor survival and cell‐cycle entry by the pre‐T‐cell receptor

Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. Although multiple reports have addressed several aspects of pre-TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR-beta locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T-cell leukemia and lymphomas. In this review, we integrate pre-TCR signaling pathways and study their effects on the regulation of T-cell progenitor cell-cycle entry and cell survival. We also connect aberrant pre-TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.

A deficiency in the in vivo clearance of apoptotic cells is a feature of the NOD mouse

Deficiencies in apoptotic cell clearance have been linked to autoimmunity. Here we examined the time-course of peritoneal macrophage phagocytosis of dying cells following the direct injection of apoptotic thymocytes into the peritoneum of NOD mice and BALB/c controls. Macrophages from NOD mice demonstrated a profound defect in the phagocytosis of apoptotic thymocytes as compared to control macrophages. Nonobese diabetic mice also demonstrated a decrease in the clearance of apoptotic cell loads following an apoptotic stimulus to thymocytes (dexamethasone) when compared to BALB/c or NOR controls. Further, NOD mice demonstrated an increase in apoptotic cell load following an apoptotic stimulus to keratinocytes (ultraviolet light, UVB) when compared to control strains. Animals deficient in macrophage phagocytosis of apoptotic debris often manifest an autoimmune phenotype characterized by the production of antinuclear autoantibodies (ANA). We determined whether increased apoptotic cell loads (through repeated exposure to UVB irradiation) could accelerate such autoimmune phenomena in young NOD mice. Following repeated UVB irradiation, NOD mice, but not BALB/c or NOR controls, developed ANA. We propose that abnormalities in apoptotic cell clearance by macrophages predispose NOD mice to autoimmunity.

The potential role of caveolin‐1 in inhibition of aquaporins during the AVD

During apoptosis, the first morphological change is a distinct cell shrinkage known as the AVD (apoptotic volume decrease). This event is driven by a loss of intracellular K(+), which creates an osmotic gradient, drawing water out of the cell through AQPs (aquaporins). Loss of water in balance with K(+) would create a shrunken cell with an equivalent intracellular concentration of K(+) ([K(+)](i) = 140 mM). However, we have previously shown that the [K(+)](i) of the shrunken apoptotic cell is 35 mM, and this level is absolutely essential for the activation of apoptotic enzymes. We have recently found that AQPs are inactivated following the AVD, so that continued loss of K(+) will reduce the intracellular concentration to this critical level. Using thymocytes, we have investigated the expression profile and regulation of the AQP family members. In the present study, we have found that AQP1, AQP8 and AQP9 are present in non-apoptotic thymocytes and localized primarily to the plasma membrane. Expression and localization did not change when these cells were induced to undergo apoptosis by growth factor withdrawal for 24 h. To explore other possible mechanisms by which these water channels are inactivated, we investigated their association with CAV-1 (caveolin-1), binding to which is known to inactivate a variety of proteins. We found that CAV-1 is present in thymocytes and that this protein co-localizes with a portion of AQP1 in normal (non-apoptotic) thymocytes. However, thymocytes induced to undergo apoptosis greatly increase their AQP1/CAV-1 association. Taken together, these results indicate that AQPs are localized to the plasma membrane of shrunken apoptotic thymocytes where increased binding to CAV-1 potentially inactivates them. AQP inactivation, coupled with continued K(+) efflux, then allows the [K(+)](i) to decrease to levels conducive for the activation of downstream apoptotic enzymes and the completion of the apoptotic cascade.

Neo-epitopes are required for immunogenicity of the La/SS-B nuclear antigen in the context of late apoptotic cells

Mechanisms responsible for the induction of anti-nuclear autoantibodies (ANA) following exposure of the immune system to an excess of apoptotic cells are incompletely understood. In this study, the immunogenicity of late apoptotic cells expressing heterologous or syngeneic forms of La/SS-B was investigated following subcutaneous administration to A/J mice, a non-autoimmune strain in which the La antigenic system is well understood. Immunization of A/J mice with late apoptotic thymocytes taken from mice transgenic (Tg) for the human La (hLa) nuclear antigen resulted in the production of IgG ANA specific for human and mouse forms of La in the absence of foreign adjuvants. Preparations of phenotypically healthy cells expressing heterologous hLa were also immunogenic. However, hLa Tg late apoptotic cells accelerated and enhanced the apparent heterologous healthy cell-induced anti-La humoral response, while non-Tg late apoptotic cells did not. Subcutaneous administration of late apoptotic cells was insufficient to break existing tolerance to the hLa antigen in hLa Tg mice or to the endogenous mouse La (mLa) antigen in A/J mice immunized with syngeneic thymocytes, indicating a requirement for the presence of heterologous epitopes for anti-La ANA production. Lymph node dendritic cells (DC) but not B cells isolated from non-Tg mice injected with hLa Tg late apoptotic cells presented immunodominant T helper cell epitopes of hLa. These studies support a model in which the generation of neo-T cell epitopes is required for loss of tolerance to nuclear proteins after exposure of the healthy immune system to an excess of cells in late stages of apoptosis.

TLX1/HOX11‐mediated disruption of primary thymocyte differentiation prior to the CD4+CD8+ double‐positive stage

The TLX1/HOX11 homeobox gene is frequently activated in T-cell acute lymphoblastic leukaemia (T-ALL) by the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations or by as yet unknown transcriptional mechanisms in the absence of 10q24 cytogenetic abnormalities. Almost all TLX1(+) T-ALLs exhibit a CD4(+)CD8(+) double-positive (DP) phenotype. To investigate the role of TLX1 as an initiating oncogene in T-ALL pathogenesis, we assessed the consequences of retroviral vector-directed TLX1 expression during the differentiation of murine and human thymocytes in fetal thymic organ cultures. Interestingly, enforced expression of TLX1 disrupted the differentiation of murine fetal liver precursors and human cord blood CD34(+) stem/progenitor cells prior to the DP thymocyte stage. Although differentiation arrest was associated with an increased percentage of apoptotic thymocytes, it could only be partially bypassed by coexpression of transgenic BCL2. Mutation of the invariant asparagine residue at position 51 of the homeodomain - which is required for efficient DNA binding - released the block, consistent with the notion that TLX1 inhibits thymocyte differentiation and promotes T-cell oncogenesis by functioning as a transcription factor. The relevance of these findings is discussed in the context of activating NOTCH1 mutations and the other genetic lesions implicated in the multistep transformation process of TLX1(+) T-ALL.

Massive Apoptosis in Lymphoid Organs in Animal Models for Primary and Secondary Progressive Multiple Sclerosis

The mechanism(s) responsible for generating the different forms of multiple sclerosis, primary progressive (PP) and secondary progressive (SP) versus relapsing-remitting (RR), is not well understood. Using myelin oligodendrocyte glycoprotein (MOG)(92-106), we have established animal models that mimic the different types of multiple sclerosis. A.SW mice develop PP or SP-experimental allergic encephalomyelitis (EAE) with large areas of demyelination and high titers of MOG antibody whereas SJL/J mice develop RR-EAE with perivascular T cells and mild demyelination. In A.SW progressive EAE, we found atrophy of the thymus, spleen, and lymph nodes with depletion of T and B cells and massive apoptosis, as demonstrated by immunohistochemistry, terminal dUTP nick-end labeling, and DNA agarose gel electrophoresis. To test whether lymphoid apoptosis itself contributes to disease progression, we injected SJL/J mice with apoptotic thymocytes. Injection of apoptotic cells resulted in greater than 20% of mice developing SP-EAE with ataxia. SJL/J mice with SP-EAE had large areas of demyelination, high MOG antibody titers and atrophic lymphoid organs. Spleen cells from mice with progressive EAE produced less interferon-gamma than those from RR-EAE when stimulated with mitogen. We suggest that induction of lymphoid apoptosis alters the balance of Th1 versus Th2 immune responses and increases MOG antibody production, leading to exacerbation of demyelination and subsequent disease progression.

Apoptotic cell clearance by phagocytes

Macrophages and dendritic cells recognize not only microorganisms foreign to the body but also dying self-cells, which they eliminate by engulfment. This phenomenon prevents dying cells from releasing potentially harmful or immunogenic intracellular materials. We found that milk fat globule-EGF-factor 8 (MFG-E8) linked apoptotic cells and phagocytes, and promoted the phagocytosis of cell corpses. MFG-E8 is expressed in tingible body macrophages (TBM) in the germinal centers of the spleen and lymph nodes. In MFG-E8-deficient mice, a large number of apoptotic lymphocytes were associated with TBM but were not engulfed, indicating that MFG-E8 was involved in the clearance of apoptotic B cells by TBM. The MFG-E8-deficient mice developed splenomegaly and produced autoantibodies such as antinuclear antibodies as well as anti-DNA antibodies in an age-dependent manner. To further examine the consequences of failure in apoptotic cell clearance, we produced a recombinant MFG-E8 mutant protein (D89E), which had inhibitory effects on the phagocytosis of apoptotic cells by a wide variety of phagocytes. When intravenously injected into mice, the D89E protein induced the production of autoantibodies. The autoantibodies production was enhanced by the coinjection of syngeneic apoptotic thymocytes. These results indicated that the impairment of apoptotic cell phagocytosis led to autoantibody production.

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.

Neutrophils Accelerate Macrophage-Mediated Digestion of Apoptotic Cells In Vivo as Well as In Vitro

It is generally believed that the clearance of apoptotic cells does not lead to inflammation. In contrast, we previously found that injection of apoptotic cells into the peritoneal cavity induced the expression of an inflammatory chemokine, MIP-2, and infiltration of neutrophils, and that anti-MIP-2 Abs suppressed the infiltration significantly. Because our previous study showed that whole-body x-irradiation caused neutrophil infiltration into the thymus along with T cell apoptosis, we examined the role of neutrophils in apoptotic cell clearance. Neutrophil infiltration reached a peak 12 h after irradiation with 1 Gy of x-rays. Immunohistological analysis revealed that apoptotic cells disappeared dramatically from 10.5 to 12 h after x-irradiation. As neutrophils moved from an inner area of the cortex to the periphery, apoptotic cells disappeared concomitantly. Either anti-MIP-2 or anti-CXCR2 Abs suppressed neutrophil infiltration significantly, and the suppression of neutrophil infiltration by anti-MIP-2 Abs delayed the disappearance of apoptotic cells. Moreover, macrophage-mediated digestion of apoptotic thymocytes was accelerated in vitro on coculturing with neutrophils, even if neutrophils were separated from macrophages. These results suggest that neutrophils are recruited to the thymus mainly by MIP-2 after whole-body x-irradiation and that such neutrophils may not induce inflammation but rather accelerate complete digestion of apoptotic cells by macrophages.

Impaired Phagocytosis in Caveolin-1 Deficient Macrophages

Caveolae are plasma membrane invaginations that function as important regulators of numerous cellular processes, including signal transduction, cholesterol trafficking, and endocytosis. Caveolin-1 (Cav-1) constitutes the main structural protein of caveolae membrane. Here, we report an in vivo increase in the number of apoptotic cells in the thymus and spleen of Cav-1 deficient mice, following whole-body γ-irradiation. We demonstrate that this increase in apoptotic cells is not due to increased apoptosis in lymphocytes per se, which normally do not express Cav-1, but rather to the decreased phagocytic clearance of apoptotic cells by macrophages, which do express Cav-1. Utilizing in vitro phagocytosis assays of both apoptotic thymocytes and Escherichia coli K-12 BioParticles, we demonstrate that the loss of Cav-1 decreases the phagocytic ability of thioglycollate-elicited peritoneal macrophages. We suggest that impaired macrophage phagocytosis in Cav-1 knockout mice could have implications for altered innate immunity against pathogens, the regulation of inflammatory responses, and the development of autoimmune disease.

Hassall's corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus

Hassall's corpuscles-first described in the human thymus over 150 years ago-are groups of epithelial cells within the thymic medulla. The physical nature of these structures differs between mammalian species. Although Hassall's corpuscles have been proposed to act in both the removal of apoptotic thymocytes and the maturation of developing thymocytes within the thymus, the function of Hassall's corpuscles has remained an enigma. Here we report that human Hassall's corpuscles express thymic stromal lymphopoietin (TSLP). Human TSLP activates thymic CD11c-positive dendritic cells to express high levels of CD80 and CD86. These TSLP-conditioned dendritic cells are then able to induce the proliferation and differentiation of CD4(+)CD8(-)CD25(-) thymic T cells into CD4(+)CD25(+)FOXP3(+) (forkhead box P3) regulatory T cells. This induction depends on peptide-major histocompatibility complex class II interactions, and the presence of CD80 and CD86, as well as interleukin 2. Immunohistochemistry studies reveal that CD25(+)CTLA4(+) (cytotoxic T-lymphocyte-associated protein 4) regulatory T cells associate in the thymic medulla with activated or mature dendritic cells and TSLP-expressing Hassall's corpuscles. These findings suggest that Hassall's corpuscles have a critical role in dendritic-cell-mediated secondary positive selection of medium-to-high affinity self-reactive T cells, leading to the generation of CD4(+)CD25(+) regulatory T cells within the thymus.

Role of Thromboxane A2in the Induction of Apoptosis of Immature Thymocytes by Lipopolysaccharide

Lipopolysaccharide (LPS) causes apoptotic deletion of CD4(+) CD8(+) thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4(+) CD8(+) thymocytes and in vitro evidence that thromboxane A(2) (TXA(2)) causes apoptosis of these cells, we tested whether enhanced generation of TXA(2) plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 micro LPS intraperitoneally displayed a marked increase in generation of TXA(2) and prostaglandin E(2) in the thymus as well as apoptotic deletion of CD4(+) CD8(+) thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA(2) mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.

Induction of apoptosis by fungal culture materials containing cyclopiazonic acid and T-2 toxin in primary lymphoid organs of broiler chickens

Thirty-six, twenty-eight-day-old broiler chicks were randomly distributed into three groups of 12 birds each. Two groups were fed diets containing 10 ppm cyclopiazonic acid (CPA) and 1ppm T-2 toxin, respectively, to determine the mechanism of cell death in spleen and thymus at 6, 12, 24, and 36 h of post-treatment. The other group served as control. T-2 toxin treated group showed significant (P < 0.01) induction of apoptosis in thymus with peak induction at 24 h post-treatment where as, no significant differences were observed between the control and CPA groups. The CPA toxin treated group showed significant (P < 0.01) induction of apoptosis in spleen with peak induction at 24 h post-treatment. No significant differences were observed between the control and T-2 toxin group even though the latter showed a slight increase in the quantity of apoptotic cells at 36 h post-treatment in spleen. The semi-thin sections stained with toluidine blue from the spleen of CPA treated group exhibited crescent margination of chromatin against the nuclear envelope and shrinkage of lymphoid cells without any surrounding inflammation, the characteristics of apoptosis. The apoptotic thymocytes from T-2 fed birds appeared shrunken with condensed nucleus and showed crescent margination of chromatin against the nuclear envelope without any surrounding inflammation when compared with well-defined nuclei with dispersed chromatin in normal thymocytes. Ultrastructurally, splenocytes of the CPA treated group and thymocytes of the T-2 toxin treated birds showed apoptotic bodies characterized by crescent margination of the chromatin against the nuclear envelope. The study indicates that one route of the CPA and T-2 toxin induced cell death in lymphoid organs of broiler chicken is by apoptosis.

Effects of PCB 126 on Primary Immune Organs and Thymocyte Apoptosis in Chicken Embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyl (PCB) 126 produce thymic atrophy and immunosuppression. This study explored the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 is associated with an increase in apoptotic thymocytes at the end of incubation in chicken embryos. Eggs were injected via the air cell with PCB 126 (0.05, 0.13, 0.32, 0.64, and 0.80 ng/g egg) on d 0 of incubation, and tissues were collected on d 20. Controls included noninjected and vehicle-injected (sunflower oil) eggs. Thymocytes were cultured for 6 h and analyzed by flow cytometry for decreased DNA content (propidium iodide staining) and cell size (forward scatter), which indicate apoptosis. PCB 126 induced dose-dependent mortality with an LD50 of 1.01 ng/g and lowest-observed-effect concentration (LOEC) of 0.32 ng/g. Teratogenic effects commonly associated with TCDD and planar PCBs, including cranial and foot deformities and subcutaneous edema, tended to increase with dose of PCB 126. PCB 126 reduced thymus mass by approximately 20% at 0.64 and 0.8 ng/g, the number of viable thymocytes by approximately 20–24% at and above 0.13 ng/g, and the number of bursal lymphoid cells by 57% at 0.64 ng/g. The percentage of apoptotic thymocytes increased with dose, reaching levels 2 times greater than controls at 0.8 ng/g. Electrophoresis of low-molecular-weight DNA from thymocytes of all doses demonstrated fragments in multiples of 180 bp. This DNA laddering is a hallmark of apoptosis. At all doses, thymocytes exhibited caspase-3 activation, another indicator of apoptosis. The results of this experiment supported the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 in chicken embryos is associated with an increase in apoptotic thymocytes on embryonic d 20.This project was supported by the U.S. Environmental Protection Agency (GR825216-01-0) and the Biomedical Sciences PhD Program at Wright State University. E. Lavoie, R. Garrett, A. Handy, and E. Reaves helped with laboratory work. Drs. Nancy Bigley, Thomas Brown, G. Allen Burton, and Robert Grubbs provided technical assistance and reviewed earlier drafts of this article.

Matrix Metalloproteinase-9 in Macrophages Induces Thymic Neovascularization following Thymocyte Apoptosis

Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.