Apoptosis in lymphoid tissues of calves inoculated with non-cytopathic bovine viral diarrhea virus genotype 1: activation of effector caspase-3 and role of macrophages

Abstract

The mechanisms responsible for lymphocyte apoptosis in bovine viral diarrhoea have not yet been clarified. Previous work suggests that bovine viral diarrhea virus (BVDV) is only directly responsible for the destruction of a small number of lymphocytes. The aim of this study was to clarify, in vivo, the role of macrophages in lymphocyte destruction through indirect mechanisms linked to the biosynthetic activation of these immunocompetent cells on ileal Peyer's patches, as well as the distribution and quantification of apoptosis. Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of BVDV genotype 1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (p.i.). The progressive depletion of Peyer's patches was found to be due to massive lymphocyte apoptosis, with an increase in cleaved caspase-3 and TUNEL-positive cells. Lymphoid depletion was accompanied, from 3 days p.i., by a significant rise in macrophage numbers both in lymphoid follicles and in interfollicular areas. Some macrophages showed signs of viral infection, together with subcellular changes indicative of phagocyte activation and, in some cases, of secretory activity. However, the number of macrophages that showed positive immunostaining for tumour necrosis factor-a and interleukin-1a, cytokines with a proven ability to induce apoptosis, remained low throughout the experiment in lymphoid follicles, where most apoptotic cells were found. These results thus appear to rule out a major involvement of macrophages and macrophage-secreted chemical mediators in the apoptosis of follicular B lymphocytes during BVDV infection. INTRODUCTIONApoptosis is a form of cell death recognized as an essential mechanism in morphogenesis and homeostasis of organs and tissues. The main morphological changes during apoptosis (cellular shrinkage, membrane blebbing, chromatin condensation at the nuclear periphery and nuclear fragmentation into apoptotic bodies) are the final result of a complex biochemical cascade of events (Huppertz et al., 1999;Rathmell & Thompson, 2002). The apoptosis cascade includes an initiation stage with induction of the cascade by external and internal stimuli, an execution stage with activation of effector proteases called caspases (cysteine-containing aspartic acid-specific proteases) and the apoptotic death stage including nuclear and cellular collapse (Huppertz et al., 1999;Hengartner, 2000). In vitro studies have elucidated two regulatory apoptosis pathways (intrinsic and extrinsic). Both pathways induce apoptosis via activation of the effector caspase-3, which, once activated, irreversibly executes cell death, so that activation of caspase-3 can be considered a hallmark of apoptosis (Stennicke et al., 1998;Huppertz et al., 1999).Apoptosis may also be involved in the immunopathogenesis of some viral diseases. There are several potential mechanisms by which viruses activate the apoptotic pathway. Some viruses may do so through the direct action of a specific viral protein (Noteborn et al., 1994;Zh...

Quantification and Determination of Spread Mechanisms of Bovine Viral Diarrhoea Virus in Blood and Tissues from Colostrum-Deprived Calves During an Experimental Acute Infection Induced by a Non-Cytopathic Genotype 1 Strain

“…Traditional methods of virus isolation quantify the amount of virus recovered from a fixed volume of blood irrespective of the cell numbers in the sample (Young et al., 2006), which during acute BVDV infection will fluctuate because of leucopoenia (Ridpath et al., 2007; Pedrera et al., 2009a; b). Thus, to quantify RNA using RT‐qPCR, controls have to be used to standardize the amounts of RNA obtained from samples from different animals taken on different days (Leutenegger et al., 2000; Young et al., 2006).…”

“…These results demonstrate the important role of lymphoid tissues in virus replication as well as the role of immunocompetent cells in the spread of BVDV throughout the body. In this sense, monocytes–macrophages and dendritic cells have been showed as the main target cells involved (Liebler‐Tenorio et al., 2003a,b, 2004; Pedrera et al., 2009a,b). The large increase in virus in the tonsils on day 9 p.i.…”

“…NCP strains, including NCP genotype 1 strains, produce mild disease in seronegative and immunocompetent infected cattle that mostly remain undetected. They are characterized by a short febrile period, transient leucopoenia and lymphoid depletion, exhibiting BVDV special tropism for lymphoid tissues (Liebler‐Tenorio et al, 2003a; b; Pedrera et al., 2009a,b).…”

Effects of Preinfection With Bovine Viral Diarrhea Virus on Immune Cells From the Lungs of Calves Inoculated With Bovine Herpesvirus 1.1

“…56 On the other hand, the increase in B-lymphocyte numbers was more marked and occurred earlier in infection in those calves inoculated with BHV-1 alone. The attenuated Blymphocyte response observed in the BVDV/BHV-1 group may be linked to a severe lymphocyte depletion in BVDV target lymphoid organs, a lesion that has already been described in calves 46 and in other in vivo infections with BVDV, 31,32,37,38,43 in which the depletion has been correlated to the action of apoptotic mechanisms developed during BVD. A difference between groups was observed in the behavior of CD8þ, CD4þ, and Foxp3þ regulatory T cells, as well as in the different cytokines studied in response to the secondary infectious agent.…”

Apoptosis in Bovine Viral Diarrhea Virus (BVDV)–Induced Mucosal Disease Lesions

“…The depletion was due to massive lymphocyte apoptosis with concurrent activation of caspase-3 as analyzed by immunohistochemistry. 30 However, lymphocyte apoptosis in the gut-associated lymphoid tissue (GALT) was paralleled by an increase in caspase-8 and rather a decrease in caspase-9, indicating that the extrinsic rather than the intrinsic pathway of apoptosis might predominate in acute BVDV infections. 29 This is in contrast to the results presented in this study that showed that the intrinsic pathway is a key element in the induction of apoptosis in MD pathogenesis in persistently infected calves.…”