- Email: [email protected]
Evolution of T Lymphocytes and Cytokine Expression in Classical Swine Fever (CSF) Virus Infection
J. Comp. Path. 2005, Vol. 132, 249–260
www.elsevier.com/locate/jcpa
Evolution of T Lymphocytes and Cytokine Expression in Classical Swine Fever (CSF) Virus Infection P. J. Sa´nchez-Cordo´n, A. Nu´n˜ez, F. J. Salguero*, L. Carrasco and J. C. Go´mez-Villamandos Departamento de Anatomı´a y Anatomı´a Patolo´gica Comparadas, Facultad de Veterinaria, Universidad de Co´rdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014 Co´rdoba and *Centro de Investigacio´n en Sanidad Animal-Instituto Nacional de Investigaciones Agrarias (CISA-INIA), Valdeolmos, Madrid, Spain
Summary This study characterized the cell-mediated immune response in pigs inoculated with the Alfort 187 isolate of classical swine fever (CSF) virus. Quantitative changes in the T-lymphocyte population (CD3C, CD4C and CD8C) and qualitative changes in cytokine expression (IL-2, IL-4 and IFNg) by these cells in serum, thymus and spleen were demonstrated. These changes coincided spatially and temporally with previously described quantitative and qualitative changes in monocyte-macrophage populations, thus demonstrating the contribution of the two cell populations to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1 cell-mediated immune response in the early and middle stages of the experiment, giving way to a type 2 immune response towards the end of the experiment; these findings, which accorded with the serological results and lymphopenia, may influence the delayed humoral response characteristic of CSF. q 2004 Elsevier Ltd. All rights reserved. Keywords: cell-mediated immunity; classical swine fever; cytokine expression; lymphoid depletion; pig; spleen; swine fever; thymus; viral infection
Introduction Macrophages, dendritic cells and natural killer cells play an important role in immunity against infectious diseases. The functions of these cells include the uptake and killing of intracellular pathogens, lysis of infected host cells, presentation of antigens to T cells and release of cytokines or chemical mediators that activate macrophages or direct the induction of T-cell subtypes. These cytokines and chemokines recruit leucocytes to the site of infection or injury, activate their antimicrobial function and regulate the induction of the adaptative response to the pathogen (McGuirk and Mills, 2000; Ryan et al., 2000). When the innate immune response fails to control the infection, the adaptative immune * Correspondence to: J.C. Go´mez-Villamandos 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2004.10.002
response is activated and this involves the production of antibodies and primed T cells. CD8C cytotoxic T lymphocytes (CTLs) kill target cells infected with viruses or bacteria, whereas CD4C T-helper (Th) cells provide help for B cells in antibody production and secrete cytokines that play a role in immunoregulatory functions or have a direct effect on invading pathogens (Germain, 1994; Jondal et al., 1996; Tizard, 1998). Thus, interferon (IFN)g and interleukin (IL)-2 are secreted during the so-called type 1 immune response, while IL-4, IL-5, IL-6, IL-10 and IL-13 are present in the type 2 immune response (Karupiah, 1998; Herna´ndez et al., 2001; McGuirk and Mills, 2002). The functions of IFNg include the stimulation of immunoglobulin production (Boehm et al., 1997; Samuel, 2001) and specific cytotoxicity of T cells (Bach et al., 1997; Biron and Sen, 2001; MacDonald q 2004 Elsevier Ltd. All rights reserved.
250
P. J. Sa´nchez-Cordo´n et al.
et al., 2002), induction of apoptosis (Tanaka et al., 1998; Samuel, 2001) and activation of resting tissue macrophages (Bach et al., 1997; Biron and Sen, 2001), thereby enhancing resistance against viral infections (Mateu de Antonio et al., 1998; Samuel, 2001). IL-2 induces growth and proliferation of T cells (Stasny et al., 2001) and stimulates their cytotoxicity (Murtaugh, 1994; Mosmann and Sad, 1996; MacDonald et al., 2002), supporting the proliferation of B cells. IL-4 promotes the development of helper and cytotoxic T cells and the differentiation of immunoglobulins-producing plasma cells from B cells (Murtaugh, 1994; Van Miert, 1995; Mosmann and Sad, 1996). The monocyte-macrophage (m-M:), identified as the main target cell for classical swine fever (CSF) virus (Summerfield et al., 1998; Go´mezVillamandos et al., 2001; Sa´nchez-Cordo´n et al., 2003), exhibited phagocytic and secretory activation leading to the synthesis and release of various chemical mediators (tumour necrosis factor [TNF]a, IL-1a and IL-6) associated with the modulation of the inflammatory and immune responses, and with the appearance of lesions characteristic of the disease (Go´mez-Villamandos et al., 2000; Bautista et al., 2002; Sa´nchez-Cordo´n et al., 2002, 2003). The cellular immune response, the characteristics of T lymphocytes and their activity against CSF virus have been studied mainly by in-vitro methods, with leucocytes isolated from peripheral blood (Pauly et al., 1995; Summerfield et al., 1996, 2001); only a few such studies have been carried out on tissue samples (Narita et al., 1996, 2000; Pauly et al., 1998). Despite the difficulty of identifying T-lymphocyte subtypes in paraffin wax-embedded, fixed tissues (Fox et al., 1985; Gonza´lez et al., 2001), such material may be useful in studying the interaction between different cells of the immune system, the presence of antigen, and the lesions. Moreover, information on immunity may be gained by demonstrating increased levels of chemical mediators, as they are directly related to the T-lymphocyte response induced by antigen (Wood and Seow, 1996; Mateu de Antonio et al., 1998). Elucidation of the mechanisms responsible for evasion of the immune response by CSF virus should lead to a better understanding of the pathogenesis of human and animal diseases caused by flaviviruses (Burke and Monath, 2001). The purpose of this CSF study was to explore the distribution and evolution of T-lymphocyte populations and the cytokines released by them in thymus and spleen, and to determine their role in pathogenesis.
Materials and Methods Animals, Virus and Experimental Design Large White x Landrace pigs (nZ36) of either sex, aged 4 months and weighing c. 30 kg were used; they were serologically negative for CSF, African swine fever, porcine reproductive and respiratory syndrome and Aujeszky’s disease. All animals were housed in the Centro de Investigacio´n en Sanidad Animal in Valdeolmos, Madrid, Spain. Thirty-two pigs each received an intramuscular inoculation of 105 50% tissue culture infective doses (TCID50) of the virulent CSF virus isolate “Alfort 187” (Wensvoort et al., 1989). The remaining four animals (controls) received only phosphate-buffered saline (PBS), pH 7.2. Clinical signs and rectal temperature were then monitored daily. The experiment was carried out in accordance with the Code of Practice for Housing and Care of Animals used in Scientific Procedures, approved by the European Economic Community Union in 1986 (86/609/EEC). Blood Collection and Enzyme-linked Immunosorbent Assay (ELISA) Preinoculation blood samples were taken from all pigs to obtain baseline values. Blood samples were taken aseptically from the anterior vena cava at 2, 3, 4, 5, 6, 7, 9, 11, 14 and 15 days post-inoculation (dpi) for leucocyte counts by means of a haemocytometer and to obtain sera for ELISA. Commercial ELISA kits were used to measure IFNg (Swine IFNg Cytoscreen; Biosource, Camarillo, CA, USA), IL-2 (Swine IL-2 CytoSets; Biosource) and IL-4 (Swine IL-4 Cytoscreen; Biosource), according to the manufacturer’s instructions. Absorbency of ELISA plates was measured by spectrophotometry (Easy Reader EAR 400; SLT-LabInstruments, Salzburg, Austria). Processing of Specimens for Structural Study and Immunohistochemistry (IHC) Infected pigs were sedated with azaperone (Stresnilw; Jannsen Animal Health, Beerese, Belgium) and killed by a overdose of thiopental-sodium (Thiovetw; Vet Limited, Leyland, Lancashire, England) in batches of four at 2, 3, 4, 7, 9, 11, 14 and 15 dpi; The remaining four pigs (controls) were killed at the end of the experiment. Samples of thymus and spleen were fixed in 10% buffered formalin solution (pH 7.2) and Bouin’s solution for structural and immunohistochemical study.
251
T Cells and Cytokine Expression in CSF
Samples were dehydrated through a graded series of alcohol, washed with xylol and embedded in paraffin wax by routine techniques for light microscopy. Wax-embedded sections (4 mm) were cut and stained with haematoxylin and eosin. The avidin–biotin-peroxidase complex (ABC) method (Hsu et al., 1981), was employed for IHC. To demonstrate CSF viral glycoprotein E2 (gp55), the monoclonal antibody WH303 was used on samples fixed in Bouin’s solution. The identification of T-lymphocyte populations and lymphocytes expressing different chemical mediators was carried out on sections (3 mm) of samples fixed in Bouin’s solution and 10% buffered formalin solution. Details of the monoclonal and polyclonal primary antibodies used, including dilutions and pre-treatments, are summarized in Table 1. PBS and non-immune serum were used in place of specific primary antibodies as negative controls. Samples from the four uninoculated animals were also used as controls. Statistical Analysis To calculate the number of immunolabelled cells present and relate the results to the different antibodies employed, two paraffin-wax blocks from thymus and spleen of each animal were selected and serial sections were used for the various immunohistochemical studies. Cells immunolabelled for viral antigen and the T cells immunolabelled by specific antibodies and expressing different chemical mediators were counted. Each cell count was made in 25 consecutive areas of 0.2 mm2 (cortex and medulla of thymus and splenic cords) and in 25 individual structures (lymphoid follicles, periarterial lymphoid sheaths and marginal zone of lymphoid follicles). The areas and structures were chosen randomly in four diagonally positioned
squares. The distribution was expressed as cells/0.2 mm2 or as cells/structure. Cells were distinguished by virtue of their location, size and morphological features. From the data on immunopositive cells, means and standard deviations (SDs) were calculated with Excel 97w (Microsoft, Washington, USA) and differences between the values in uninoculated and inoculated animals were tested for significance (P%0.05) by Student’s t-test.
Results Clinical Signs and Gross Lesions Control animals remained healthy. The clinical signs of CSF observed in infected animals from 2 dpi consisted of persistent pyrexia (40.5–41.5 8C), varying degrees of anorexia, and ocular discharges associated with conjunctivitis. From 6 dpi onwards body temperatures rose to O42 8C, and animals showed decreased activity and semi-liquid yellowish-grey diarrhoea. From 2 dpi, hyperaemia and petechiae were seen in submandibular lymph nodes and tonsils. From 4 dpi pigs showed erythema of the skin, petechial and ecchymotic haemorrhages in the lung, and necrosis in the tonsils. Some showed petechiae in the kidney and gall bladder, and ileal congestion, with diarrhoea and melena. Reduction in size of the thymus was observed from 7 dpi onwards, together with occasional splenic infarcts characteristic of CSF. From 9 dpi, animals developed the typical staggering gait associated with CSF and a cyanotic discoloration of the skin of the abdomen, snout, ears and legs. Leucocyte Counts and Cytokines in Sera Leucocyte counts were described by Sa´nchez-Cordo´n et al. (2002). Briefly, total leucocytes in
Table 1 Details of the immunolabelling reagents Primary antibody 1
WH303, monoclonal CD3, polyclonal2 CD4, monoclonal3 CD8, monoclonal3 IL-2, polyclonal4 IL-4, polyclonal4 IFNg, polyclonal5
Fixative
Antigen or cell detected
Antibody dilution*
Pretreatment
Bouin’s solution Buffered formalin solution 10% Bouin’s solution Bouin’s solution Bouin’s solution Bouin’s solution Bouin’s solution
Viral glycoprotein gp-55 Human T-lymphocytes
1 in 100 1 in 500
None or TC† Protease‡
Anti-porcine CD4 T-cells Anti-porcine CD8 T-cells Anti-porcine IL-2 Anti-porcine IL-4 Anti-porcine IFNg
1 in 10 1 in 10 1 in 50 1 in 50 1 in 200
TC† TC† Tween 20§ Tween 20§ TC†
* In PBS, pH 7.2, containing normal goat serum 10%. The avidin–biotin complex (ABC) method (Vector Laboratories, Burlingame, CA, USA) was used with 3,3 0 diaminobenzidine tetrahydrochloride (Sigma-Aldrich Chemie, Steinheim, Germany) as chromogen. †(TC)ZTrisodium citrate dihydrate (Merck, Mu¨nchen, Germany), 0.01 molar (pH 3.2), microwave 10 min. §Tweenw 20 (Merck) 0.1%, 10 min. ‡ Protease type XIV (Sigma-Aldrich Chemie) 0.1%, 10 min. 1Veterinary Laboratories Agency, Addlestone, UK. 2Dako, Glostrup, Denmark. 3 BioVet-UCO, Co´rdoba, Spain. 4Biosource, Camarillo, CA, USA. 5Endogen, Woburn, MA, USA.
252
P. J. Sa´nchez-Cordo´n et al.
disappeared. In the spleen, from 7 dpi, an increase in m-M: numbers in splenic cords, lymphoid structures (mainly follicles) and the marginal zone coincided with intense lymphoid depletion, a decrease in the size of follicles, and the disappearance of germinal centres. The evidence of apoptosis was previously studied and confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique and by transmission electron microscopy (Sa´nchez-Cordo´n et al., 2002, 2003). Fig. 1 Means of the concentrations of IL-2 ($), IL-4 (&) and IFNg (6) in sera (pg/ml) of uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint).
blood, especially lymphocytes, showed a significant increase at 2 dpi. However, from 4 dpi to the end of the experiment they decreased significantly. The concentration of IL-2 increased significantly at 2 dpi (Fig. 1) but thereafter decreased to a concentration no different from that of the control animals. IL-4 and IFNg peaked at 7 dpi and decreased thereafter. Histopathology From 2–3 dpi, inoculated animals showed hypocellularity in the thymic cortex and in splenic lymphoid structures, particularly lymphoid follicles, which remained unchanged in size. This lymphocyte depletion was accompanied by pyknosis and karyorrhexis, suggestive of apoptosis. The onset of apoptosis coincided, from 2 dpi onwards, with an increase in the size and number of m-M:s; these contained engulfed cell debris, giving rise to so-called “tingible body” macrophages. From 4 dpi, the cortex assumed a “starry sky” appearance; it was also smaller, and by 9 dpi had completely
Viral Antigen The control pigs showed no immunolabelling for the glycoprotein E2 (gp55) of CSF virus. In infected animals, however, viral antigen was detected from 2 dpi in thymus and spleen, mainly in m-M:s and occasionally in fibroblasts and star-shaped reticuloepithelial cells. Only occasional infected lymphocytes were detected at 4 dpi, but from 7 dpi there was a marked increase in the number of infected cells, mainly m-M:s, in the thymic cortex and medulla and in all splenic compartments. At this time, the number of infected lymphocytes, polymorphonuclear neutrophils and endothelial cells was slightly increased in both organs. The increase in number of infected cells peaked at 9 and 11 dpi in thymic medulla and splenic structures, respectively, declining thereafter. Quantitative Changes in T-Lymphocyte Populations From 4 dpi, there was a significant increase, peaking at 7 dpi, in mature CD3C lymphocytes in the thymic cortex as compared with the controls (Fig. 2A). Subsequently, atrophy of the cortex prevented further lymphocyte counts. Almost all
Fig. 2 A,B. (A) Count (meanGSD) of CD3C lymphocytes (&) per area (0.2 mm2) of thymic cortex in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls. (B) Count (meanGSD) of CD3C lymphocytes per area (0.2 mm2) of splenic cords (:) and per individual lymphoid structure (follicles [&], marginal zone of lymphoid follicles [,] and periarterial lymphoid sheaths [ ]), in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
T Cells and Cytokine Expression in CSF
medullary lymphocytes, in both experimental and control animals, were CD3C. In the spleen (Fig. 2B), a significant increase in mature CD3C lymphocytes was observed in peripheral areas of follicles from 2 dpi, lasting until 11 dpi. Periarterial lymphoid sheaths showed a significant fall in numbers of mature CD3C lymphocytes; no positive cells were observed in these areas at 14 and 15 dpi. There was no significant change in CD3C lymphocyte numbers in the marginal zone until 9 dpi, which marked the start of a significant decline that lasted until the end of the experiment. The number of CD3C lymphocytes in splenic cords was significantly lower in experimental animals than in controls, except at 9 dpi; thereafter, numbers recovered until the end of the experiment. No CD8C cells were observed in the thymic cortex or spleen of untreated controls. There was a notable absence of CD4C and CD8C cells in the periarterial lymphoid sheaths of infected animals. Despite this, from 2 dpi there was a significant increase in the number of mature CD4C and CD8C T lymphocytes (Fig. 3A) in both thymic regions, lasting until the end of the experiment (Fig. 4A and B). During the early stages of the disease (i.e. up to 7 dpi), mature CD4C T lymphocytes predominated in both cortex and medulla. Thenceforth, however, they were outnumbered by mature CD8C T lymphocytes. From 11 dpi, both subpopulations showed a further increase in immunolabelled medullary lymphocytes, lasting until the end of the experiment; CD4C and CD8C T lymphocytes, which peaked at 15 dpi, showed a focal, predominantly perivascular arrangement (Fig. 3A). The number of mature CD4C T lymphocytes was significantly greater than in controls at 2 dpi (Fig. 5A, C, and E), in splenic cords and follicles; thereafter, the difference ceased to be significant until 7 dpi in follicles, where they peaked at 14 dpi, and in splenic cords. In the marginal zone, however, immunolabelled cell numbers showed no significant changes. CD4C T lymphocytes were widely dispersed in all splenic compartments; from 11 dpi, positive cell clusters were also visible in certain lymphoid structures (Fig. 3B). At 2 dpi, CD8C T lymphocytes, usually widespread, showed a significant increase in the splenic compartments examined; a further increase was observed from 9 dpi, peaking at 14 dpi in follicles and in the marginal zone, and at 15 dpi in splenic cords.
253
Cytokine Expression by T Lymphocytes There were more IL-2-positive than IL-4-positive lymphocytes in both thymic regions (Fig. 4C and D); no IL-4-positive cells were observed in the cortex of untreated controls. From 2 dpi until the end of the experiment, the number of cells immunolabelled for both mediators was significantly greater than in controls, in both cortex and medulla. In the early stages of the disease, the number of IL-2-positive lymphocytes peaked at 3 dpi in the cortex and medulla. Subsequently, a further increase was recorded in the medulla from 11 dpi, peak values occurring at 15 dpi. IL-2positive lymphocytes were observed throughout the cortex, close to capillaries. The numbers of IL-4-positive lymphocytes showed no statistically significant differences from those of the controls, except for a peak recorded at 3 dpi in the cortex and an increase from 14 dpi onwards in the medulla. In the cortex, immunolabelled cells tended to be highly dispersed, but occasional clusters of positive cells occurred, particularly in the vicinity of blood vessels (Fig. 3C). From 2 dpi to the end of the experiment, there was a significant increase in IL-2- and IL-4-positive lymphocytes in splenic cords and follicular structures (Fig. 5B, D, and F); up to 9 dpi (but with the exception of 4 dpi), however, IL-2-positive lymphocytes outnumbered IL-4-positive lymphocytes in these compartments. Between 11 and 14 dpi, there was a slight increase in the number of cells immunolabelled for both interleukins. When numbers peaked, there was a predominance of IL-4-positive lymphocytes that lasted until the end of the experiment. By contrast, IL-positive cell numbers in the marginal zone were not significantly increased except at 4, 7 and 11 dpi, when an increase was noted in IL-4-positive lymphocytes. Positive cells were generally scattered through spleen compartments (Fig. 3D). No IL-2- or IL-4-positive lymphocytes were observed in periarterial lymphoid sheaths. The numbers of lymphocytes immunolabelled for IFNg rose above those of the controls from 2 dpi in the cortex and 3 dpi in the medulla (Fig. 6A), reaching maximum values at 7 dpi and 11 dpi, respectively. Towards the end of the experiment (14–15 dpi), the numbers of IFNgpositive lymphocytes fell well below those of earlier peaks, although remaining significantly higher than in the untreated controls. Throughout the experiment, IFNg-positive lymphocyte numbers increased significantly above those of the controls, in all splenic compartments
254
P. J. Sa´nchez-Cordo´n et al.
Fig. 3 A–F. (A) CD8C T lymphocytes in medulla of thymus at 15 dpi. IHC. Bar, 80 mm. (B) Detection of CD4C T lymphocytes at 11 dpi. Note the immunolabelled cells, mainly in follicle and in the marginal zone of spleen. IHC. Bar, 80 mm. (C) Lymphocytes immunolabelled for IL-2 in the cortex of the thymus at 3 dpi. Note the hypocellularity caused by the lymphoid depletion. IHC. Bar, 80 mm. (D) IL-4-positive lymphocytes in follicle of spleen at 14 dpi. IHC. Bar, 40 mm. (E,F) IFNg-positive lymphocytes in splenic lymphoid follicles at 3 dpi (E) and in splenic cords at 9 dpi (F). Note the progressive lymphoid depletion. IHC. Bars, 80 mm. AR, artery; F, follicle; HC, Hassall’s corpuscle; MZ, marginal zone; PLS, periarterial lymphoid sheath; SC, splenic cords; T, trabeculae.
T Cells and Cytokine Expression in CSF
255
Fig. 4 A–D. Counts (meanGSD) of CD4C (&) and CD8C (,) T lymphocytes and lymphocytes immunolabelled against IL-2 ( ) and IL-4 ( ) in cortex (A, C) and medulla (B, D) of the thymus in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
(Fig. 6B); this increase was apparent in the marginal zone and within follicles from 2 dpi, and in splenic cords from 3 dpi (Fig. 3E). Periarterial lymphoid sheaths showed an increase in immunolabelled lymphocytes only at 7 and 9 dpi (Fig. 3F). During the early stages of the disease, the numbers of IFNg-positive lymphocytes rose at 3 dpi, declining slightly thereafter to a non-significant level until 9 dpi, when the maximum numbers were recorded in all splenic compartments. Numbers then gradually declined until the end of the experiment, although remaining significantly higher than in untreated controls (except in periarterial lymphoid sheaths).
Discussion This study characterized the cell-mediated immune response in CSF, demonstrating quantitative changes in the T-lymphocyte population and qualitative changes in cytokine expression by these cells in serum, thymus and spleen; these changes coincided spatially and temporally with quantitative and qualitative changes in mM: populations (Go´mez-Villamandos et al., 2001; Sa´nchez-Cordo´n et al., 2002, 2003). Thus, both cell populations contribute to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1
cell-mediated immune response in the early and middle stages of the experiment, giving way later to a type 2 immune response; these findings, which accorded with the serological results and lymphopenia, may help to explain the delayed humoral response characteristic of CSF. The lymphoid depletion habitually found in CSF is caused by lymphocyte apoptosis linked to the release by m-M:s of certain chemical mediators (Sa´ nchez-Cordo´n et al., 2002). Despite this depletion, observed in the thymic cortex in CSF, the present study revealed an increase in the number of cells immunolabelled for the CD3 complex, which is present only in mature T cells (Kearse et al., 1995; Arnaud et al., 1996; Roitt et al., 1998). This would suggest that the increase reflected enhanced T-lymphocyte activation signal transfer (Straus and Weiss, 1993) as a response to antigen-presenting cells (m-M:s) (Brown et al., 1993; Roitt et al., 1998), which infiltrated the thymic cortex and showed changes in secretory activation from 2 dpi onwards (Sa´nchez-Cordo´n et al., 2002). These findings suggest that differentiation and maturation of T lymphocytes in the thymic cortex are potentiated in CSF, although T lymphocyte apoptosis impairs the effectiveness of the nonspecific immune response. In the spleen, however, CD3C T lymphocyte numbers declined, possibly due to apoptosis;
256
P. J. Sa´nchez-Cordo´n et al.
Fig. 5 A–F. Counts (meanGSD) of CD4C (&) and CD8C (,) T lymphocytes and lymphocytes immunolabelled against IL-2 ( ) and IL-4 ( ) per area (0.2 mm2) of splenic cords (A, B) and per individual lymphoid structure (marginal zone of lymphoid follicles [C, D] and follicles [E, F]) in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
Fig. 6 A,B. (A) Counts (meanGSD) of lymphocytes immunolabelled against IFNg in cortex (&) and medulla ( ) of thymus in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls. (B) Counts (meanGSD) of lymphocytes immunolabelled against IFNg per area (0.2 mm2) of splenic cords (:) and per individual lymphoid structure (follicles [&], marginal zone of lymphoid follicles [ ] and periarterial lymphoid sheaths [,]) in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
T Cells and Cytokine Expression in CSF
the decline was most marked in the middle stage of the disease (9 dpi), a stage in which the thymic cortex was found to be totally atrophied (Sato et al., 2000; Sa´nchez-Cordo´n et al., 2002). The non-arrival of T lymphocytes from the thymic cortex might prevent any recovery of T-lymphocyte populations in the spleen. Even so, a slight recovery of CD3C T lymphocytes was observed in splenic cords from 9 dpi, at which point numbers were significantly higher than in controls; thereafter, though falling significantly, numbers remained higher than at the start of the disease, possibly due to T-cell clonal expansion (Tizard, 1998), prompted by the high concentrations of viral antigen detected in this splenic compartment. From the onset of the disease in the present study, an increase was noted in the number of CD4C and CD8C lymphocytes (Narita et al., 1996, 2000), and CD8C outnumbered CD4C T cells at advanced stages (Narita et al., 1996, 2000; Pauly et al., 1998; Lee et al., 1999). This increase, despite intense lymphoid depletion, may have been due to activation and differentiation of lymphocytes still remaining in the organs examined, which may have played a role in inducing a cell-mediated response to the virus (Ober et al., 1998; Herna´ndez et al., 2001). The behaviour of lymphocyte populations may be mediated by the proliferative and secretory changes displayed by m-M:s in the thymus and the spleen. Increased synthesis and release of IL-1 would account for the early increase in CD4C T lymphocyte numbers, which ceased when the presence of this mediator declined (Komai-Koma et al., 2003). Increase in the number of CD8C T cells is more closely linked to expression of IL-6 (Van Snick, 1990; Sa´nchez-Cordo´n et al., 2002), whose appearance and increase took place later in the present study and may have been related to the clonal expansion mentioned above (Tizard, 1998). CD8C T cells are cytotoxic for CSF virus, and their increase during the disease may therefore be part of a defence mechanism (Doherty et al., 1992; Pauly et al., 1995; Summerfield et al., 1996); this was demonstrated when vaccinated swine remained protected from the virus despite the absence of neutralizing antibodies (Ru¨menapf et al., 1991; Suradhat et al., 2001). Depending on the chemical mediators predominating in the course of a disease, the cellmediated immune response may be classified as type 1 or type 2 (Karupiah, 1998; Herna´ndez et al., 2001); to date, however, the cell-mediated response in CSF has not been characterized. The results of the present study point to increased production of IL-2 and IFNg in the early and middle stages of
257
the disease, suggesting a type 1 immune response. The increased number of macrophages functioning as antigen-presenting cells probably prompted the activation of IL-2-producing T lymphocytes; IL-2 would in turn promote the proliferation of B lymphocytes (Murtaugh, 1994; Mosmann and Sad, 1996). Synthesis of IL-2 may be enhanced by macrophage secretion of IL-1 and IL-6, with IFNg inducing an autocrine effect on the production of this cytokine (Biron and Sen, 2001). This would account for the two-phase behaviour of IL-2 observed here, the first being associated with the presence of viral antigen, and the second with high IFNg concentrations. IL-4 suppresses macrophage secretion of TNFa, IL-1 and IL-6 (Roitt et al., 1998); increased IL-4 concentrations may therefore account for the low concentrations of these cytokines observed in the final stages of CSF (Sa´nchez-Cordo´n et al., 2002). Moreover, given its ability to induce generation of CD4C CD8C T cells (Paliard et al., 1988; Brod et al., 1990), IL-4 may have played a part in the numerical increase in CD8C T cells towards the end of the experiment. Similarly, the presence of IL-4-positive lymphocytes may be related to the start of delayed production of virus-specific antibodies (Ru¨menapf et al., 1991; Laevens et al., 1999; Suradhat et al., 2001), since IL-4 induces the division of B cells and their differentiation into plasma cells (Murtaugh, 1994; Mosmann and Sad, 1996). IFNg, secreted by activated T lymphocytes (Luster et al., 1999), is considered to be the main activator of resting tissue macrophages, increasing their phagocytic and secretory capacity (Collart et al., 1986; Bach et al., 1997; Biron and Sen, 2001); such activation may have taken place from the start of the present experiment, in which the behaviour of IFNg-expressing cells mirrored that of TNFa, IL-1a and IL-6 expression by m-M:s, suggesting a potent synergism between these cytokines, also reported in other swine viral infections (Van Reeth et al., 1998). Although to a lesser extent than other interferons, IFNg displays antiviral activity (Samuel, 1991, 1998; Biron and Sen, 2001), derived from its ability to induce apoptosis (Tanaka et al., 1998; Samuel, 2001) and from the chemotactics of cell defence; a number of authors have highlighted its protective role in CSF (Suradhat et al., 2001). The results obtained here suggest that this activity has a harmful effect on the animal by inducing apoptosis in uninfected defence cells and promoting the spread of virus. It is therefore likely that IFNg induces release of TNFa and IL-1a by m-M:s, with a consequent increase in cytotoxic capacity
258
P. J. Sa´nchez-Cordo´n et al.
(Bach et al., 1997; Biron and Sen, 2001); this may be a major factor in inducing the lymphocyte apoptosis associated with these chemical mediators in the course of CSF (Sa´nchez-Cordo´n et al., 2002). Early apoptosis of infected m-M:s and phagocytosis of apoptotic bodies by other m-M:s may play a decisive role in the spread of the virus and in its initial evasion of the immune response (Go´mez-Villamandos et al., 2001). IFNg may be a key element in this process, not only because of its ability to induce direct apoptosis of infected cells, but also because it exerts an indirect chemotactic effect through IL-1, enhancing the recruitment of m-M:s and increasing the number of infectable target cells. This would account for the increase in m-M: numbers observed in the course of CSF (Go´mez-Villamandos et al., 2001; Sa´nchez-Cordo´n et al., 2002); it would also explain why the maximum expression of IFNg coincided with the maximum presence of infected cells in both spleen and thymus.
Acknowledgments This work was supported by grants from DGESICMEC (PB98-1033) and Junta de Andalucı´a (Plan Andaluz de Investigacio´n AGR-0137). We appreciate the technical assistance of “Servicio Central de Apoyo a la Investigacio´n (SCAI), Co´rdoba University”. P.J. Sa´nchez-Cordo´n is recipient of a fellowship from MCYT (Plan MIT).
References Arnaud, J., Chenu, C., Huchenq, A., Gouaillard, C., Kuhlmann, J. and Rubin, B. (1996). Defective interactions between TCR chains and CD3 heterodimers prevent membrane expression of TCRab in human T cells. Journal of Immunology, 156, 2155– 2162. Bach, E. A., Aguet, M. and Schreiber, R. D. (1997). The interferon gamma receptor: a paradigm for cytokine receptor signalling. Annual Review of Immunology, 15, 563–591. Bautista, M. J., Ruiz-Villamor, E., Salguero, F. J., Sa´nchezCordo´n, P. J., Carrasco, L. and Go´mez-Villamandos, J. C. (2002). Early platelet aggregation as a cause of thrombocytopenia in classical swine fever. Veterinary Pathology, 39, 84–91. Biron, C. A. and Sen, G. C. (2001). Interferon and other cytokines. In: Fields Virology, 4th Edit., D. M. Knipe, P. M. Howley, D. E. Griffin, M. Martin, B. Roizman and S. E. Straus, Eds, Lippincott Williams and Wilkins, Philadelphia, pp. 321–351.
Boehm, U., Klamp, T., Groot, M. and Howard, J. C. (1997). Cellular responses to interferon-gamma. Annual Review of Immunology, 15, 749–795. Brod, S. A., Purvee, M., Benjamin, P. and Hafler, D. A. (1990). Frequency analysis of CD4C CD8C T-cells cloned with IL-4. Cellular Immunology, 175, 426–436. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Stromiger, J. L. and Wiley, D. C. (1993). Three-dimensional structure of the human class II antigen HLA-DR1. Nature, 364, 33–39. Burke, D. S. and Monath, T. P. (2001). Flaviviruses. In: Fields Virology, 4th Edit., P. M. Knipe, P. M. Howley, D. E. Griffin, M. Martin, B. Roizman and S. E. Straus, Eds, Lippincott Williams and Wilkins, Philadelphia, pp. 1043–1125. Collart, M. A., Belin, D., Vassalli, J. D., de Kossodo, S. and Vassalli, P. (1986). Gamma interferon enhances macrophage transcription of the tumour necrosis factor/cachectin, interleukin 1 and urokinase genes, which are controlled by short-lived repressors. Journal of Experimental Medicine, 164, 2113– 2118. Doherty, P. C., Allan, W. and Eichelberger, M. (1992). Roles of ab and gd T cell subsets in viral immunity. Annual Review of Immunology, 10, 123–151. Fox, C. H., Johnson, F. B., Whiting, J. and Roller, P. P. (1985). Formaldehyde fixation. Journal of Histochemistry and Cytochemistry, 33, 845–853. Germain, R. N. (1994). MCH-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell, 76, 287–299. Go´mez-Villamandos, J. C., Ruiz-Villamor, E., Bautista, M. J., Quezada, M., Sa´nchez, C. P., Salguero, F. J. and Sierra, M. A. (2000). Pathogenesis of classical swine fever: renal haemorrhages and erythrodiapedesis. Journal of Comparative Pathology, 123, 47– 54. Go´mez-Villamandos, J. C., Ruiz-Villamor, E., Bautista, M. J., Sa´nchez, C. P., Sa´nchez-Cordo´n, P. J., Salguero, F. J. and Jover, A. (2001). Morphological and immunohistochemical changes in splenic macrophages of pigs infected with classical swine fever. Journal of Comparative Pathology, 125, 98–109. Gonza´lez, L., Anderson, I., Deane, D., Summers, C. and Buxton, D. (2001). Detection of immune system cells in paraffin wax-embedded ovine tissues. Journal of Comparative Pathology, 125, 41–47. Herna´ndez, J., Garfias, Y., Nieto, A., Mercado, C., Montan ˜ o, L. F. and Centeno, E. (2001). Comparative evaluation of CD4CCD8C and CD4CCD8K lymphocytes in the immune response to porcine rubulavirus. Veterinary Immunology and Immunopathology, 79, 249–259. Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin–biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry, 29, 577–580.
T Cells and Cytokine Expression in CSF
Jondal, M., Schirmbeck, R. and Reimann, J. (1996). MCH class I-restricted CTL responses to exogenous antigens. Immunity, 5, 295–302. Karupiah, G. (1998). Type 1 and Type 2 cytokines in antiviral defense. Veterinary Immunology and Immunopathology, 63, 105–109. Kearse, K. P., Roberts, J. P. and Singer, A. (1995). TCRaCD3d3 association is the initial step in ab dimmer formation in murine T cells and is limiting in immature CD3CCD8C thymocytes. Immunity, 2, 391–399. Komai-Koma, M., Gracie, J. A., Wei, X. Q., Xu, D., Thomson, N., McInnes, I. B. and Liew, F. Y. (2003). Chemoattraction of human T cells by IL-18. Journal of Immunology, 170, 1084–1090. Laevens, H., Koenen, F., Deluyker, H. and de Kruif, A. (1999). Experimental infection of slaughter pigs with classical swine fever virus: transmission of the virus, course of the disease and antibody response. Veterinary Record, 145, 243–248. Lee, W. C., Wang, C. S. and Chien, M. S. (1999). Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection. Veterinary Microbiology, 67, 17–29. Luster, M. I., Simeonova, P. P., Gallucci, R. and Matheson, J. (1999). Tumor necrosis factor a and toxicology. Critical Reviews in Toxicology, 29, 491–511. MacDonald, A. J., Duffy, M., Brady, M. T., McKiernan, S., Hall, W., Hegarty, J., Curry, M. and Mills, K. H. G. (2002). CD4C T helper type 1 and T regulatory type 2 cells induced against the same epitopes on the core protein in hepatitis C virus-infected individuals. Journal of Infectious Diseases, 185, 720–727. Mateu de Antonio, E., Husmann, R. J., Hansen, R., Lunney, J. K., Strom, D., Martı´n, S. and Zuckermann, F. A. (1998). Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen. Veterinary Immunology and Immunopathology, 61, 265–277. McGuirk, P. and Mills, K. H. G. (2000). Direct antiinflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous haemagglutinin from Bordetella pertussis. European Journal of Immunology, 30, 415–422. McGuirk, P. and Mills, K. H. G. (2002). Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends in Immunology, 23, 450–455. Mosmann, T. R. and Sad, S. (1996). The expanding universe of T-cell subsets: Th1, Th2 and more. Immunology Today, 17, 138–146. Murtaugh, M. P. (1994). Porcine cytokines. Veterinary Immunology and Immunopathology, 43, 37–44. Narita, M., Kawashima, K., Kimura, K., Mikami, O., Shibahara, T., Yamada, S. and Sakoda, Y. (2000). Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus. Veterinary Pathology, 37, 402–408.
259
Narita, M., Kawashima, K. and Shimizu, M. (1996). Viral antigen and B and T lymphocytes in lymphoid tissues of gnotobiotic piglets infected with hog cholera virus. Journal of Comparative Pathology, 114, 257–263. Ober, B. T., Summerfield, A., Mattlinger, C., Wiesmuller, K. H., Jung, G., Pfaff, E., Saalmu¨ller, A. and Rziha, H. J. (1998). Vaccine-induced pseudorabies virusspecific, extrathymic CD4CCD8C memory T-helper cells in swine. Journal of Virology, 72, 4866–4873. Paliard, X., Malefijt, R. W., de Vries, J. E. and Spits, H. (1988). IL-4 mediates CD8 induction on human CD4C T-cell clones. Nature, 355, 642–644. Pauly, T., Elbers, K., Ko¨ nig, M., Lengsfeld, T., Saalmu¨ller, A. and Thiel, H. J. (1995). Classical swine fever virus-specific cytotoxic T lymphocytes and identification of a T cell epitope. Journal of General Virology, 76, 3039–3049. Pauly, T., Ko¨nig, M., Thiel, H. J. and Saalmu¨ller, A. (1998). Infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine T lymphocytes. Journal of General Virology, 79, 31–40. Roitt, I., Brostoff, J. and Male, D. (1998). Receptores de las ce´lulas T y mole´culas del CPH. In: Inmunologı´a, 4th Edit., I. Roitt, J. Brostoff and D. Male, Eds, Harcourt Brace, Madrid, pp. 5.1–5.9. Ru¨menapf, T., Stark, R., Meyers, G. and Thiel, H. J. (1991). Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. Journal of Virology, 65, 589–597. Ryan, E. J., McNeela, E., Pizza, M., Rappuoli, R., O’Neil, L. and Mill, K. H. G. (2000). Modulation of innate and acquired immune response by Escherichia coli heat labile toxin: distinct pro- and anti-inflammatory effects of the non-toxic AB complex and the enzyme activity. Journal of Immunology, 165, 5750–5759. Samuel, C. E. (1991). Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology, 183, 1–11. Samuel, C. E. (1998). Reoviruses and the interferon system. Current Topics in Microbiology and Immunology, 233, 125–145. Samuel, C. E. (2001). Antiviral action of interferons. Clinical Microbiology Reviews, 14, 778–809. Sa´nchez-Cordo´n, P. J., Romanini, S., Salguero, F. J., Nu´n ˜ ez, A., Bautista, M. J., Jover, A. and Go´mezVillamandos, J. C. (2002). Apoptosis of thymocytes related to cytokine expression in experimental classical swine fever. Journal of Comparative Pathology, 127, 239–248. Sa´nchez-Cordo´n, P. J., Romanini, S., Salguero, F. J., RuizVillamor, E., Carrasco, L. and Go´mez-Villamandos, J. C. (2003). A histopathological, immunohistochemical and ultrastructural study of the intestine in pigs inoculated with classical swine fever virus. Veterinary Pathology, 40, 254–262.
260
P. J. Sa´nchez-Cordo´n et al.
Sato, M., Mikami, O., Kobayashi, M. and Nakajima, Y. (2000). Apoptosis in the lymphatic organs of piglets inoculated with classical swine fever virus. Veterinary Microbiology, 75, 1–9. Stasny, B. R., de Guise, S., Rompato, G. and Garmendia, A. E. (2001). Functional characterization of a swine CD4C/CD8C double positive lymphoblastoid T-cell line with a CD25C/CD45RAK phenotype generated in vitro with IL-2. Veterinary Immunology and Immunopathology, 78, 57–70. Straus, D. B. and Weiss, A. (1993). The CD3 chains of the T cell antigen receptor associated with the ZAP-70 tyrosine kinase are tyrosine phosphorylated after receptor stimulation. Journal of Experimental Medicine, 178, 1523–1530. Summerfield, A., Kno¨tig, S. M. and McCullough, K. C. (1998). Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. Journal of Virology, 72, 1853–1861. Summerfield, A., McNeilly, F., Walker, I., Allan, G., Knoeting, S. M. and McCullough, K. C. (2001). Depletion of CD4C and CD8highC T-cells before the onset of viraemia during classical swine fever. Veterinary Immunology and Immunopathology, 78, 3–19. Summerfield, A., Rziha, H. J. and Saalmu¨ller, A. (1996). Functional characterization of porcine CD4CCD8C extrathymic T lymphocytes. Cellular Immunology, 168, 291–296. Suradhat, S., Intrakamhaeng, M. and Damrongwatanapokin, S. (2001). The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection. Veterinary Immunology and Immunopathology, 83, 177–189.
Tanaka, N., Sato, M., Lamphier, M., Nozawa, H., Oda, E., Noguchi, S., Schreiber, R. D., Tsujimoto, Y. and Taniguchi, T. (1998). Type 1 interferons are essential mediators of apoptotic death in virally infected cells. Genes Cells, 3, 29–37. Tizard, I. R. (1998). Ce´lulas T auxiliaries y su respuesta al antı´geno. In: Inmunologı´a Veterinaria, 5th Edit., I. R. Tizard, Ed., McGraw-Hill Interamericana Editores, Me´xico, pp. 119–131. Van Miert, A. (1995). Pro-inflammatory cytokines in a ruminant model: pathophysiological, pharmacological, and therapeutic aspects. Veterinary Quarterly, 175, 41–50. Van Reeth, K., Nauwynck, H. and Pensaert, M. (1998). Production of interferon alpha, tumor necrosis factor alpha, interleukin-1 and inflammation during acute influenza in pigs: a possible model for humans? Journal of Infectious Diseases, 177, 1076–1079. Van Snick, J. (1990). Interleukin-6: an overview. Annual Review of Immunology, 8, 253–278. Wensvoort, G., Terpstra, C., de Kluijver, E. P., Kragten, C. and Warnaar, J. C. (1989). Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Veterinary Microbiology, 21, 9–20. Wood, P. R. and Seow, H. F. (1996). T cell cytokines and disease prevention. Veterinary Immunology and Immunopathology, 54, 33–34.
Received; June 7th; 2004 Accepted; October 18th; 2004
www.elsevier.com/locate/jcpa
Evolution of T Lymphocytes and Cytokine Expression in Classical Swine Fever (CSF) Virus Infection P. J. Sa´nchez-Cordo´n, A. Nu´n˜ez, F. J. Salguero*, L. Carrasco and J. C. Go´mez-Villamandos Departamento de Anatomı´a y Anatomı´a Patolo´gica Comparadas, Facultad de Veterinaria, Universidad de Co´rdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014 Co´rdoba and *Centro de Investigacio´n en Sanidad Animal-Instituto Nacional de Investigaciones Agrarias (CISA-INIA), Valdeolmos, Madrid, Spain
Summary This study characterized the cell-mediated immune response in pigs inoculated with the Alfort 187 isolate of classical swine fever (CSF) virus. Quantitative changes in the T-lymphocyte population (CD3C, CD4C and CD8C) and qualitative changes in cytokine expression (IL-2, IL-4 and IFNg) by these cells in serum, thymus and spleen were demonstrated. These changes coincided spatially and temporally with previously described quantitative and qualitative changes in monocyte-macrophage populations, thus demonstrating the contribution of the two cell populations to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1 cell-mediated immune response in the early and middle stages of the experiment, giving way to a type 2 immune response towards the end of the experiment; these findings, which accorded with the serological results and lymphopenia, may influence the delayed humoral response characteristic of CSF. q 2004 Elsevier Ltd. All rights reserved. Keywords: cell-mediated immunity; classical swine fever; cytokine expression; lymphoid depletion; pig; spleen; swine fever; thymus; viral infection
Introduction Macrophages, dendritic cells and natural killer cells play an important role in immunity against infectious diseases. The functions of these cells include the uptake and killing of intracellular pathogens, lysis of infected host cells, presentation of antigens to T cells and release of cytokines or chemical mediators that activate macrophages or direct the induction of T-cell subtypes. These cytokines and chemokines recruit leucocytes to the site of infection or injury, activate their antimicrobial function and regulate the induction of the adaptative response to the pathogen (McGuirk and Mills, 2000; Ryan et al., 2000). When the innate immune response fails to control the infection, the adaptative immune * Correspondence to: J.C. Go´mez-Villamandos 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2004.10.002
response is activated and this involves the production of antibodies and primed T cells. CD8C cytotoxic T lymphocytes (CTLs) kill target cells infected with viruses or bacteria, whereas CD4C T-helper (Th) cells provide help for B cells in antibody production and secrete cytokines that play a role in immunoregulatory functions or have a direct effect on invading pathogens (Germain, 1994; Jondal et al., 1996; Tizard, 1998). Thus, interferon (IFN)g and interleukin (IL)-2 are secreted during the so-called type 1 immune response, while IL-4, IL-5, IL-6, IL-10 and IL-13 are present in the type 2 immune response (Karupiah, 1998; Herna´ndez et al., 2001; McGuirk and Mills, 2002). The functions of IFNg include the stimulation of immunoglobulin production (Boehm et al., 1997; Samuel, 2001) and specific cytotoxicity of T cells (Bach et al., 1997; Biron and Sen, 2001; MacDonald q 2004 Elsevier Ltd. All rights reserved.
250
P. J. Sa´nchez-Cordo´n et al.
et al., 2002), induction of apoptosis (Tanaka et al., 1998; Samuel, 2001) and activation of resting tissue macrophages (Bach et al., 1997; Biron and Sen, 2001), thereby enhancing resistance against viral infections (Mateu de Antonio et al., 1998; Samuel, 2001). IL-2 induces growth and proliferation of T cells (Stasny et al., 2001) and stimulates their cytotoxicity (Murtaugh, 1994; Mosmann and Sad, 1996; MacDonald et al., 2002), supporting the proliferation of B cells. IL-4 promotes the development of helper and cytotoxic T cells and the differentiation of immunoglobulins-producing plasma cells from B cells (Murtaugh, 1994; Van Miert, 1995; Mosmann and Sad, 1996). The monocyte-macrophage (m-M:), identified as the main target cell for classical swine fever (CSF) virus (Summerfield et al., 1998; Go´mezVillamandos et al., 2001; Sa´nchez-Cordo´n et al., 2003), exhibited phagocytic and secretory activation leading to the synthesis and release of various chemical mediators (tumour necrosis factor [TNF]a, IL-1a and IL-6) associated with the modulation of the inflammatory and immune responses, and with the appearance of lesions characteristic of the disease (Go´mez-Villamandos et al., 2000; Bautista et al., 2002; Sa´nchez-Cordo´n et al., 2002, 2003). The cellular immune response, the characteristics of T lymphocytes and their activity against CSF virus have been studied mainly by in-vitro methods, with leucocytes isolated from peripheral blood (Pauly et al., 1995; Summerfield et al., 1996, 2001); only a few such studies have been carried out on tissue samples (Narita et al., 1996, 2000; Pauly et al., 1998). Despite the difficulty of identifying T-lymphocyte subtypes in paraffin wax-embedded, fixed tissues (Fox et al., 1985; Gonza´lez et al., 2001), such material may be useful in studying the interaction between different cells of the immune system, the presence of antigen, and the lesions. Moreover, information on immunity may be gained by demonstrating increased levels of chemical mediators, as they are directly related to the T-lymphocyte response induced by antigen (Wood and Seow, 1996; Mateu de Antonio et al., 1998). Elucidation of the mechanisms responsible for evasion of the immune response by CSF virus should lead to a better understanding of the pathogenesis of human and animal diseases caused by flaviviruses (Burke and Monath, 2001). The purpose of this CSF study was to explore the distribution and evolution of T-lymphocyte populations and the cytokines released by them in thymus and spleen, and to determine their role in pathogenesis.
Materials and Methods Animals, Virus and Experimental Design Large White x Landrace pigs (nZ36) of either sex, aged 4 months and weighing c. 30 kg were used; they were serologically negative for CSF, African swine fever, porcine reproductive and respiratory syndrome and Aujeszky’s disease. All animals were housed in the Centro de Investigacio´n en Sanidad Animal in Valdeolmos, Madrid, Spain. Thirty-two pigs each received an intramuscular inoculation of 105 50% tissue culture infective doses (TCID50) of the virulent CSF virus isolate “Alfort 187” (Wensvoort et al., 1989). The remaining four animals (controls) received only phosphate-buffered saline (PBS), pH 7.2. Clinical signs and rectal temperature were then monitored daily. The experiment was carried out in accordance with the Code of Practice for Housing and Care of Animals used in Scientific Procedures, approved by the European Economic Community Union in 1986 (86/609/EEC). Blood Collection and Enzyme-linked Immunosorbent Assay (ELISA) Preinoculation blood samples were taken from all pigs to obtain baseline values. Blood samples were taken aseptically from the anterior vena cava at 2, 3, 4, 5, 6, 7, 9, 11, 14 and 15 days post-inoculation (dpi) for leucocyte counts by means of a haemocytometer and to obtain sera for ELISA. Commercial ELISA kits were used to measure IFNg (Swine IFNg Cytoscreen; Biosource, Camarillo, CA, USA), IL-2 (Swine IL-2 CytoSets; Biosource) and IL-4 (Swine IL-4 Cytoscreen; Biosource), according to the manufacturer’s instructions. Absorbency of ELISA plates was measured by spectrophotometry (Easy Reader EAR 400; SLT-LabInstruments, Salzburg, Austria). Processing of Specimens for Structural Study and Immunohistochemistry (IHC) Infected pigs were sedated with azaperone (Stresnilw; Jannsen Animal Health, Beerese, Belgium) and killed by a overdose of thiopental-sodium (Thiovetw; Vet Limited, Leyland, Lancashire, England) in batches of four at 2, 3, 4, 7, 9, 11, 14 and 15 dpi; The remaining four pigs (controls) were killed at the end of the experiment. Samples of thymus and spleen were fixed in 10% buffered formalin solution (pH 7.2) and Bouin’s solution for structural and immunohistochemical study.
251
T Cells and Cytokine Expression in CSF
Samples were dehydrated through a graded series of alcohol, washed with xylol and embedded in paraffin wax by routine techniques for light microscopy. Wax-embedded sections (4 mm) were cut and stained with haematoxylin and eosin. The avidin–biotin-peroxidase complex (ABC) method (Hsu et al., 1981), was employed for IHC. To demonstrate CSF viral glycoprotein E2 (gp55), the monoclonal antibody WH303 was used on samples fixed in Bouin’s solution. The identification of T-lymphocyte populations and lymphocytes expressing different chemical mediators was carried out on sections (3 mm) of samples fixed in Bouin’s solution and 10% buffered formalin solution. Details of the monoclonal and polyclonal primary antibodies used, including dilutions and pre-treatments, are summarized in Table 1. PBS and non-immune serum were used in place of specific primary antibodies as negative controls. Samples from the four uninoculated animals were also used as controls. Statistical Analysis To calculate the number of immunolabelled cells present and relate the results to the different antibodies employed, two paraffin-wax blocks from thymus and spleen of each animal were selected and serial sections were used for the various immunohistochemical studies. Cells immunolabelled for viral antigen and the T cells immunolabelled by specific antibodies and expressing different chemical mediators were counted. Each cell count was made in 25 consecutive areas of 0.2 mm2 (cortex and medulla of thymus and splenic cords) and in 25 individual structures (lymphoid follicles, periarterial lymphoid sheaths and marginal zone of lymphoid follicles). The areas and structures were chosen randomly in four diagonally positioned
squares. The distribution was expressed as cells/0.2 mm2 or as cells/structure. Cells were distinguished by virtue of their location, size and morphological features. From the data on immunopositive cells, means and standard deviations (SDs) were calculated with Excel 97w (Microsoft, Washington, USA) and differences between the values in uninoculated and inoculated animals were tested for significance (P%0.05) by Student’s t-test.
Results Clinical Signs and Gross Lesions Control animals remained healthy. The clinical signs of CSF observed in infected animals from 2 dpi consisted of persistent pyrexia (40.5–41.5 8C), varying degrees of anorexia, and ocular discharges associated with conjunctivitis. From 6 dpi onwards body temperatures rose to O42 8C, and animals showed decreased activity and semi-liquid yellowish-grey diarrhoea. From 2 dpi, hyperaemia and petechiae were seen in submandibular lymph nodes and tonsils. From 4 dpi pigs showed erythema of the skin, petechial and ecchymotic haemorrhages in the lung, and necrosis in the tonsils. Some showed petechiae in the kidney and gall bladder, and ileal congestion, with diarrhoea and melena. Reduction in size of the thymus was observed from 7 dpi onwards, together with occasional splenic infarcts characteristic of CSF. From 9 dpi, animals developed the typical staggering gait associated with CSF and a cyanotic discoloration of the skin of the abdomen, snout, ears and legs. Leucocyte Counts and Cytokines in Sera Leucocyte counts were described by Sa´nchez-Cordo´n et al. (2002). Briefly, total leucocytes in
Table 1 Details of the immunolabelling reagents Primary antibody 1
WH303, monoclonal CD3, polyclonal2 CD4, monoclonal3 CD8, monoclonal3 IL-2, polyclonal4 IL-4, polyclonal4 IFNg, polyclonal5
Fixative
Antigen or cell detected
Antibody dilution*
Pretreatment
Bouin’s solution Buffered formalin solution 10% Bouin’s solution Bouin’s solution Bouin’s solution Bouin’s solution Bouin’s solution
Viral glycoprotein gp-55 Human T-lymphocytes
1 in 100 1 in 500
None or TC† Protease‡
Anti-porcine CD4 T-cells Anti-porcine CD8 T-cells Anti-porcine IL-2 Anti-porcine IL-4 Anti-porcine IFNg
1 in 10 1 in 10 1 in 50 1 in 50 1 in 200
TC† TC† Tween 20§ Tween 20§ TC†
* In PBS, pH 7.2, containing normal goat serum 10%. The avidin–biotin complex (ABC) method (Vector Laboratories, Burlingame, CA, USA) was used with 3,3 0 diaminobenzidine tetrahydrochloride (Sigma-Aldrich Chemie, Steinheim, Germany) as chromogen. †(TC)ZTrisodium citrate dihydrate (Merck, Mu¨nchen, Germany), 0.01 molar (pH 3.2), microwave 10 min. §Tweenw 20 (Merck) 0.1%, 10 min. ‡ Protease type XIV (Sigma-Aldrich Chemie) 0.1%, 10 min. 1Veterinary Laboratories Agency, Addlestone, UK. 2Dako, Glostrup, Denmark. 3 BioVet-UCO, Co´rdoba, Spain. 4Biosource, Camarillo, CA, USA. 5Endogen, Woburn, MA, USA.
252
P. J. Sa´nchez-Cordo´n et al.
disappeared. In the spleen, from 7 dpi, an increase in m-M: numbers in splenic cords, lymphoid structures (mainly follicles) and the marginal zone coincided with intense lymphoid depletion, a decrease in the size of follicles, and the disappearance of germinal centres. The evidence of apoptosis was previously studied and confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique and by transmission electron microscopy (Sa´nchez-Cordo´n et al., 2002, 2003). Fig. 1 Means of the concentrations of IL-2 ($), IL-4 (&) and IFNg (6) in sera (pg/ml) of uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint).
blood, especially lymphocytes, showed a significant increase at 2 dpi. However, from 4 dpi to the end of the experiment they decreased significantly. The concentration of IL-2 increased significantly at 2 dpi (Fig. 1) but thereafter decreased to a concentration no different from that of the control animals. IL-4 and IFNg peaked at 7 dpi and decreased thereafter. Histopathology From 2–3 dpi, inoculated animals showed hypocellularity in the thymic cortex and in splenic lymphoid structures, particularly lymphoid follicles, which remained unchanged in size. This lymphocyte depletion was accompanied by pyknosis and karyorrhexis, suggestive of apoptosis. The onset of apoptosis coincided, from 2 dpi onwards, with an increase in the size and number of m-M:s; these contained engulfed cell debris, giving rise to so-called “tingible body” macrophages. From 4 dpi, the cortex assumed a “starry sky” appearance; it was also smaller, and by 9 dpi had completely
Viral Antigen The control pigs showed no immunolabelling for the glycoprotein E2 (gp55) of CSF virus. In infected animals, however, viral antigen was detected from 2 dpi in thymus and spleen, mainly in m-M:s and occasionally in fibroblasts and star-shaped reticuloepithelial cells. Only occasional infected lymphocytes were detected at 4 dpi, but from 7 dpi there was a marked increase in the number of infected cells, mainly m-M:s, in the thymic cortex and medulla and in all splenic compartments. At this time, the number of infected lymphocytes, polymorphonuclear neutrophils and endothelial cells was slightly increased in both organs. The increase in number of infected cells peaked at 9 and 11 dpi in thymic medulla and splenic structures, respectively, declining thereafter. Quantitative Changes in T-Lymphocyte Populations From 4 dpi, there was a significant increase, peaking at 7 dpi, in mature CD3C lymphocytes in the thymic cortex as compared with the controls (Fig. 2A). Subsequently, atrophy of the cortex prevented further lymphocyte counts. Almost all
Fig. 2 A,B. (A) Count (meanGSD) of CD3C lymphocytes (&) per area (0.2 mm2) of thymic cortex in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls. (B) Count (meanGSD) of CD3C lymphocytes per area (0.2 mm2) of splenic cords (:) and per individual lymphoid structure (follicles [&], marginal zone of lymphoid follicles [,] and periarterial lymphoid sheaths [ ]), in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
T Cells and Cytokine Expression in CSF
medullary lymphocytes, in both experimental and control animals, were CD3C. In the spleen (Fig. 2B), a significant increase in mature CD3C lymphocytes was observed in peripheral areas of follicles from 2 dpi, lasting until 11 dpi. Periarterial lymphoid sheaths showed a significant fall in numbers of mature CD3C lymphocytes; no positive cells were observed in these areas at 14 and 15 dpi. There was no significant change in CD3C lymphocyte numbers in the marginal zone until 9 dpi, which marked the start of a significant decline that lasted until the end of the experiment. The number of CD3C lymphocytes in splenic cords was significantly lower in experimental animals than in controls, except at 9 dpi; thereafter, numbers recovered until the end of the experiment. No CD8C cells were observed in the thymic cortex or spleen of untreated controls. There was a notable absence of CD4C and CD8C cells in the periarterial lymphoid sheaths of infected animals. Despite this, from 2 dpi there was a significant increase in the number of mature CD4C and CD8C T lymphocytes (Fig. 3A) in both thymic regions, lasting until the end of the experiment (Fig. 4A and B). During the early stages of the disease (i.e. up to 7 dpi), mature CD4C T lymphocytes predominated in both cortex and medulla. Thenceforth, however, they were outnumbered by mature CD8C T lymphocytes. From 11 dpi, both subpopulations showed a further increase in immunolabelled medullary lymphocytes, lasting until the end of the experiment; CD4C and CD8C T lymphocytes, which peaked at 15 dpi, showed a focal, predominantly perivascular arrangement (Fig. 3A). The number of mature CD4C T lymphocytes was significantly greater than in controls at 2 dpi (Fig. 5A, C, and E), in splenic cords and follicles; thereafter, the difference ceased to be significant until 7 dpi in follicles, where they peaked at 14 dpi, and in splenic cords. In the marginal zone, however, immunolabelled cell numbers showed no significant changes. CD4C T lymphocytes were widely dispersed in all splenic compartments; from 11 dpi, positive cell clusters were also visible in certain lymphoid structures (Fig. 3B). At 2 dpi, CD8C T lymphocytes, usually widespread, showed a significant increase in the splenic compartments examined; a further increase was observed from 9 dpi, peaking at 14 dpi in follicles and in the marginal zone, and at 15 dpi in splenic cords.
253
Cytokine Expression by T Lymphocytes There were more IL-2-positive than IL-4-positive lymphocytes in both thymic regions (Fig. 4C and D); no IL-4-positive cells were observed in the cortex of untreated controls. From 2 dpi until the end of the experiment, the number of cells immunolabelled for both mediators was significantly greater than in controls, in both cortex and medulla. In the early stages of the disease, the number of IL-2-positive lymphocytes peaked at 3 dpi in the cortex and medulla. Subsequently, a further increase was recorded in the medulla from 11 dpi, peak values occurring at 15 dpi. IL-2positive lymphocytes were observed throughout the cortex, close to capillaries. The numbers of IL-4-positive lymphocytes showed no statistically significant differences from those of the controls, except for a peak recorded at 3 dpi in the cortex and an increase from 14 dpi onwards in the medulla. In the cortex, immunolabelled cells tended to be highly dispersed, but occasional clusters of positive cells occurred, particularly in the vicinity of blood vessels (Fig. 3C). From 2 dpi to the end of the experiment, there was a significant increase in IL-2- and IL-4-positive lymphocytes in splenic cords and follicular structures (Fig. 5B, D, and F); up to 9 dpi (but with the exception of 4 dpi), however, IL-2-positive lymphocytes outnumbered IL-4-positive lymphocytes in these compartments. Between 11 and 14 dpi, there was a slight increase in the number of cells immunolabelled for both interleukins. When numbers peaked, there was a predominance of IL-4-positive lymphocytes that lasted until the end of the experiment. By contrast, IL-positive cell numbers in the marginal zone were not significantly increased except at 4, 7 and 11 dpi, when an increase was noted in IL-4-positive lymphocytes. Positive cells were generally scattered through spleen compartments (Fig. 3D). No IL-2- or IL-4-positive lymphocytes were observed in periarterial lymphoid sheaths. The numbers of lymphocytes immunolabelled for IFNg rose above those of the controls from 2 dpi in the cortex and 3 dpi in the medulla (Fig. 6A), reaching maximum values at 7 dpi and 11 dpi, respectively. Towards the end of the experiment (14–15 dpi), the numbers of IFNgpositive lymphocytes fell well below those of earlier peaks, although remaining significantly higher than in the untreated controls. Throughout the experiment, IFNg-positive lymphocyte numbers increased significantly above those of the controls, in all splenic compartments
254
P. J. Sa´nchez-Cordo´n et al.
Fig. 3 A–F. (A) CD8C T lymphocytes in medulla of thymus at 15 dpi. IHC. Bar, 80 mm. (B) Detection of CD4C T lymphocytes at 11 dpi. Note the immunolabelled cells, mainly in follicle and in the marginal zone of spleen. IHC. Bar, 80 mm. (C) Lymphocytes immunolabelled for IL-2 in the cortex of the thymus at 3 dpi. Note the hypocellularity caused by the lymphoid depletion. IHC. Bar, 80 mm. (D) IL-4-positive lymphocytes in follicle of spleen at 14 dpi. IHC. Bar, 40 mm. (E,F) IFNg-positive lymphocytes in splenic lymphoid follicles at 3 dpi (E) and in splenic cords at 9 dpi (F). Note the progressive lymphoid depletion. IHC. Bars, 80 mm. AR, artery; F, follicle; HC, Hassall’s corpuscle; MZ, marginal zone; PLS, periarterial lymphoid sheath; SC, splenic cords; T, trabeculae.
T Cells and Cytokine Expression in CSF
255
Fig. 4 A–D. Counts (meanGSD) of CD4C (&) and CD8C (,) T lymphocytes and lymphocytes immunolabelled against IL-2 ( ) and IL-4 ( ) in cortex (A, C) and medulla (B, D) of the thymus in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
(Fig. 6B); this increase was apparent in the marginal zone and within follicles from 2 dpi, and in splenic cords from 3 dpi (Fig. 3E). Periarterial lymphoid sheaths showed an increase in immunolabelled lymphocytes only at 7 and 9 dpi (Fig. 3F). During the early stages of the disease, the numbers of IFNg-positive lymphocytes rose at 3 dpi, declining slightly thereafter to a non-significant level until 9 dpi, when the maximum numbers were recorded in all splenic compartments. Numbers then gradually declined until the end of the experiment, although remaining significantly higher than in untreated controls (except in periarterial lymphoid sheaths).
Discussion This study characterized the cell-mediated immune response in CSF, demonstrating quantitative changes in the T-lymphocyte population and qualitative changes in cytokine expression by these cells in serum, thymus and spleen; these changes coincided spatially and temporally with quantitative and qualitative changes in mM: populations (Go´mez-Villamandos et al., 2001; Sa´nchez-Cordo´n et al., 2002, 2003). Thus, both cell populations contribute to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1
cell-mediated immune response in the early and middle stages of the experiment, giving way later to a type 2 immune response; these findings, which accorded with the serological results and lymphopenia, may help to explain the delayed humoral response characteristic of CSF. The lymphoid depletion habitually found in CSF is caused by lymphocyte apoptosis linked to the release by m-M:s of certain chemical mediators (Sa´ nchez-Cordo´n et al., 2002). Despite this depletion, observed in the thymic cortex in CSF, the present study revealed an increase in the number of cells immunolabelled for the CD3 complex, which is present only in mature T cells (Kearse et al., 1995; Arnaud et al., 1996; Roitt et al., 1998). This would suggest that the increase reflected enhanced T-lymphocyte activation signal transfer (Straus and Weiss, 1993) as a response to antigen-presenting cells (m-M:s) (Brown et al., 1993; Roitt et al., 1998), which infiltrated the thymic cortex and showed changes in secretory activation from 2 dpi onwards (Sa´nchez-Cordo´n et al., 2002). These findings suggest that differentiation and maturation of T lymphocytes in the thymic cortex are potentiated in CSF, although T lymphocyte apoptosis impairs the effectiveness of the nonspecific immune response. In the spleen, however, CD3C T lymphocyte numbers declined, possibly due to apoptosis;
256
P. J. Sa´nchez-Cordo´n et al.
Fig. 5 A–F. Counts (meanGSD) of CD4C (&) and CD8C (,) T lymphocytes and lymphocytes immunolabelled against IL-2 ( ) and IL-4 ( ) per area (0.2 mm2) of splenic cords (A, B) and per individual lymphoid structure (marginal zone of lymphoid follicles [C, D] and follicles [E, F]) in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
Fig. 6 A,B. (A) Counts (meanGSD) of lymphocytes immunolabelled against IFNg in cortex (&) and medulla ( ) of thymus in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls. (B) Counts (meanGSD) of lymphocytes immunolabelled against IFNg per area (0.2 mm2) of splenic cords (:) and per individual lymphoid structure (follicles [&], marginal zone of lymphoid follicles [ ] and periarterial lymphoid sheaths [,]) in uninoculated (UI) pigs (nZ4) and pigs inoculated with CSF virus (nZ4 pigs per timepoint). *Significantly different (P%0.05) from controls.
T Cells and Cytokine Expression in CSF
the decline was most marked in the middle stage of the disease (9 dpi), a stage in which the thymic cortex was found to be totally atrophied (Sato et al., 2000; Sa´nchez-Cordo´n et al., 2002). The non-arrival of T lymphocytes from the thymic cortex might prevent any recovery of T-lymphocyte populations in the spleen. Even so, a slight recovery of CD3C T lymphocytes was observed in splenic cords from 9 dpi, at which point numbers were significantly higher than in controls; thereafter, though falling significantly, numbers remained higher than at the start of the disease, possibly due to T-cell clonal expansion (Tizard, 1998), prompted by the high concentrations of viral antigen detected in this splenic compartment. From the onset of the disease in the present study, an increase was noted in the number of CD4C and CD8C lymphocytes (Narita et al., 1996, 2000), and CD8C outnumbered CD4C T cells at advanced stages (Narita et al., 1996, 2000; Pauly et al., 1998; Lee et al., 1999). This increase, despite intense lymphoid depletion, may have been due to activation and differentiation of lymphocytes still remaining in the organs examined, which may have played a role in inducing a cell-mediated response to the virus (Ober et al., 1998; Herna´ndez et al., 2001). The behaviour of lymphocyte populations may be mediated by the proliferative and secretory changes displayed by m-M:s in the thymus and the spleen. Increased synthesis and release of IL-1 would account for the early increase in CD4C T lymphocyte numbers, which ceased when the presence of this mediator declined (Komai-Koma et al., 2003). Increase in the number of CD8C T cells is more closely linked to expression of IL-6 (Van Snick, 1990; Sa´nchez-Cordo´n et al., 2002), whose appearance and increase took place later in the present study and may have been related to the clonal expansion mentioned above (Tizard, 1998). CD8C T cells are cytotoxic for CSF virus, and their increase during the disease may therefore be part of a defence mechanism (Doherty et al., 1992; Pauly et al., 1995; Summerfield et al., 1996); this was demonstrated when vaccinated swine remained protected from the virus despite the absence of neutralizing antibodies (Ru¨menapf et al., 1991; Suradhat et al., 2001). Depending on the chemical mediators predominating in the course of a disease, the cellmediated immune response may be classified as type 1 or type 2 (Karupiah, 1998; Herna´ndez et al., 2001); to date, however, the cell-mediated response in CSF has not been characterized. The results of the present study point to increased production of IL-2 and IFNg in the early and middle stages of
257
the disease, suggesting a type 1 immune response. The increased number of macrophages functioning as antigen-presenting cells probably prompted the activation of IL-2-producing T lymphocytes; IL-2 would in turn promote the proliferation of B lymphocytes (Murtaugh, 1994; Mosmann and Sad, 1996). Synthesis of IL-2 may be enhanced by macrophage secretion of IL-1 and IL-6, with IFNg inducing an autocrine effect on the production of this cytokine (Biron and Sen, 2001). This would account for the two-phase behaviour of IL-2 observed here, the first being associated with the presence of viral antigen, and the second with high IFNg concentrations. IL-4 suppresses macrophage secretion of TNFa, IL-1 and IL-6 (Roitt et al., 1998); increased IL-4 concentrations may therefore account for the low concentrations of these cytokines observed in the final stages of CSF (Sa´nchez-Cordo´n et al., 2002). Moreover, given its ability to induce generation of CD4C CD8C T cells (Paliard et al., 1988; Brod et al., 1990), IL-4 may have played a part in the numerical increase in CD8C T cells towards the end of the experiment. Similarly, the presence of IL-4-positive lymphocytes may be related to the start of delayed production of virus-specific antibodies (Ru¨menapf et al., 1991; Laevens et al., 1999; Suradhat et al., 2001), since IL-4 induces the division of B cells and their differentiation into plasma cells (Murtaugh, 1994; Mosmann and Sad, 1996). IFNg, secreted by activated T lymphocytes (Luster et al., 1999), is considered to be the main activator of resting tissue macrophages, increasing their phagocytic and secretory capacity (Collart et al., 1986; Bach et al., 1997; Biron and Sen, 2001); such activation may have taken place from the start of the present experiment, in which the behaviour of IFNg-expressing cells mirrored that of TNFa, IL-1a and IL-6 expression by m-M:s, suggesting a potent synergism between these cytokines, also reported in other swine viral infections (Van Reeth et al., 1998). Although to a lesser extent than other interferons, IFNg displays antiviral activity (Samuel, 1991, 1998; Biron and Sen, 2001), derived from its ability to induce apoptosis (Tanaka et al., 1998; Samuel, 2001) and from the chemotactics of cell defence; a number of authors have highlighted its protective role in CSF (Suradhat et al., 2001). The results obtained here suggest that this activity has a harmful effect on the animal by inducing apoptosis in uninfected defence cells and promoting the spread of virus. It is therefore likely that IFNg induces release of TNFa and IL-1a by m-M:s, with a consequent increase in cytotoxic capacity
258
P. J. Sa´nchez-Cordo´n et al.
(Bach et al., 1997; Biron and Sen, 2001); this may be a major factor in inducing the lymphocyte apoptosis associated with these chemical mediators in the course of CSF (Sa´nchez-Cordo´n et al., 2002). Early apoptosis of infected m-M:s and phagocytosis of apoptotic bodies by other m-M:s may play a decisive role in the spread of the virus and in its initial evasion of the immune response (Go´mez-Villamandos et al., 2001). IFNg may be a key element in this process, not only because of its ability to induce direct apoptosis of infected cells, but also because it exerts an indirect chemotactic effect through IL-1, enhancing the recruitment of m-M:s and increasing the number of infectable target cells. This would account for the increase in m-M: numbers observed in the course of CSF (Go´mez-Villamandos et al., 2001; Sa´nchez-Cordo´n et al., 2002); it would also explain why the maximum expression of IFNg coincided with the maximum presence of infected cells in both spleen and thymus.
Acknowledgments This work was supported by grants from DGESICMEC (PB98-1033) and Junta de Andalucı´a (Plan Andaluz de Investigacio´n AGR-0137). We appreciate the technical assistance of “Servicio Central de Apoyo a la Investigacio´n (SCAI), Co´rdoba University”. P.J. Sa´nchez-Cordo´n is recipient of a fellowship from MCYT (Plan MIT).
References Arnaud, J., Chenu, C., Huchenq, A., Gouaillard, C., Kuhlmann, J. and Rubin, B. (1996). Defective interactions between TCR chains and CD3 heterodimers prevent membrane expression of TCRab in human T cells. Journal of Immunology, 156, 2155– 2162. Bach, E. A., Aguet, M. and Schreiber, R. D. (1997). The interferon gamma receptor: a paradigm for cytokine receptor signalling. Annual Review of Immunology, 15, 563–591. Bautista, M. J., Ruiz-Villamor, E., Salguero, F. J., Sa´nchezCordo´n, P. J., Carrasco, L. and Go´mez-Villamandos, J. C. (2002). Early platelet aggregation as a cause of thrombocytopenia in classical swine fever. Veterinary Pathology, 39, 84–91. Biron, C. A. and Sen, G. C. (2001). Interferon and other cytokines. In: Fields Virology, 4th Edit., D. M. Knipe, P. M. Howley, D. E. Griffin, M. Martin, B. Roizman and S. E. Straus, Eds, Lippincott Williams and Wilkins, Philadelphia, pp. 321–351.
Boehm, U., Klamp, T., Groot, M. and Howard, J. C. (1997). Cellular responses to interferon-gamma. Annual Review of Immunology, 15, 749–795. Brod, S. A., Purvee, M., Benjamin, P. and Hafler, D. A. (1990). Frequency analysis of CD4C CD8C T-cells cloned with IL-4. Cellular Immunology, 175, 426–436. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Stromiger, J. L. and Wiley, D. C. (1993). Three-dimensional structure of the human class II antigen HLA-DR1. Nature, 364, 33–39. Burke, D. S. and Monath, T. P. (2001). Flaviviruses. In: Fields Virology, 4th Edit., P. M. Knipe, P. M. Howley, D. E. Griffin, M. Martin, B. Roizman and S. E. Straus, Eds, Lippincott Williams and Wilkins, Philadelphia, pp. 1043–1125. Collart, M. A., Belin, D., Vassalli, J. D., de Kossodo, S. and Vassalli, P. (1986). Gamma interferon enhances macrophage transcription of the tumour necrosis factor/cachectin, interleukin 1 and urokinase genes, which are controlled by short-lived repressors. Journal of Experimental Medicine, 164, 2113– 2118. Doherty, P. C., Allan, W. and Eichelberger, M. (1992). Roles of ab and gd T cell subsets in viral immunity. Annual Review of Immunology, 10, 123–151. Fox, C. H., Johnson, F. B., Whiting, J. and Roller, P. P. (1985). Formaldehyde fixation. Journal of Histochemistry and Cytochemistry, 33, 845–853. Germain, R. N. (1994). MCH-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell, 76, 287–299. Go´mez-Villamandos, J. C., Ruiz-Villamor, E., Bautista, M. J., Quezada, M., Sa´nchez, C. P., Salguero, F. J. and Sierra, M. A. (2000). Pathogenesis of classical swine fever: renal haemorrhages and erythrodiapedesis. Journal of Comparative Pathology, 123, 47– 54. Go´mez-Villamandos, J. C., Ruiz-Villamor, E., Bautista, M. J., Sa´nchez, C. P., Sa´nchez-Cordo´n, P. J., Salguero, F. J. and Jover, A. (2001). Morphological and immunohistochemical changes in splenic macrophages of pigs infected with classical swine fever. Journal of Comparative Pathology, 125, 98–109. Gonza´lez, L., Anderson, I., Deane, D., Summers, C. and Buxton, D. (2001). Detection of immune system cells in paraffin wax-embedded ovine tissues. Journal of Comparative Pathology, 125, 41–47. Herna´ndez, J., Garfias, Y., Nieto, A., Mercado, C., Montan ˜ o, L. F. and Centeno, E. (2001). Comparative evaluation of CD4CCD8C and CD4CCD8K lymphocytes in the immune response to porcine rubulavirus. Veterinary Immunology and Immunopathology, 79, 249–259. Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin–biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry, 29, 577–580.
T Cells and Cytokine Expression in CSF
Jondal, M., Schirmbeck, R. and Reimann, J. (1996). MCH class I-restricted CTL responses to exogenous antigens. Immunity, 5, 295–302. Karupiah, G. (1998). Type 1 and Type 2 cytokines in antiviral defense. Veterinary Immunology and Immunopathology, 63, 105–109. Kearse, K. P., Roberts, J. P. and Singer, A. (1995). TCRaCD3d3 association is the initial step in ab dimmer formation in murine T cells and is limiting in immature CD3CCD8C thymocytes. Immunity, 2, 391–399. Komai-Koma, M., Gracie, J. A., Wei, X. Q., Xu, D., Thomson, N., McInnes, I. B. and Liew, F. Y. (2003). Chemoattraction of human T cells by IL-18. Journal of Immunology, 170, 1084–1090. Laevens, H., Koenen, F., Deluyker, H. and de Kruif, A. (1999). Experimental infection of slaughter pigs with classical swine fever virus: transmission of the virus, course of the disease and antibody response. Veterinary Record, 145, 243–248. Lee, W. C., Wang, C. S. and Chien, M. S. (1999). Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection. Veterinary Microbiology, 67, 17–29. Luster, M. I., Simeonova, P. P., Gallucci, R. and Matheson, J. (1999). Tumor necrosis factor a and toxicology. Critical Reviews in Toxicology, 29, 491–511. MacDonald, A. J., Duffy, M., Brady, M. T., McKiernan, S., Hall, W., Hegarty, J., Curry, M. and Mills, K. H. G. (2002). CD4C T helper type 1 and T regulatory type 2 cells induced against the same epitopes on the core protein in hepatitis C virus-infected individuals. Journal of Infectious Diseases, 185, 720–727. Mateu de Antonio, E., Husmann, R. J., Hansen, R., Lunney, J. K., Strom, D., Martı´n, S. and Zuckermann, F. A. (1998). Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen. Veterinary Immunology and Immunopathology, 61, 265–277. McGuirk, P. and Mills, K. H. G. (2000). Direct antiinflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous haemagglutinin from Bordetella pertussis. European Journal of Immunology, 30, 415–422. McGuirk, P. and Mills, K. H. G. (2002). Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends in Immunology, 23, 450–455. Mosmann, T. R. and Sad, S. (1996). The expanding universe of T-cell subsets: Th1, Th2 and more. Immunology Today, 17, 138–146. Murtaugh, M. P. (1994). Porcine cytokines. Veterinary Immunology and Immunopathology, 43, 37–44. Narita, M., Kawashima, K., Kimura, K., Mikami, O., Shibahara, T., Yamada, S. and Sakoda, Y. (2000). Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus. Veterinary Pathology, 37, 402–408.
259
Narita, M., Kawashima, K. and Shimizu, M. (1996). Viral antigen and B and T lymphocytes in lymphoid tissues of gnotobiotic piglets infected with hog cholera virus. Journal of Comparative Pathology, 114, 257–263. Ober, B. T., Summerfield, A., Mattlinger, C., Wiesmuller, K. H., Jung, G., Pfaff, E., Saalmu¨ller, A. and Rziha, H. J. (1998). Vaccine-induced pseudorabies virusspecific, extrathymic CD4CCD8C memory T-helper cells in swine. Journal of Virology, 72, 4866–4873. Paliard, X., Malefijt, R. W., de Vries, J. E. and Spits, H. (1988). IL-4 mediates CD8 induction on human CD4C T-cell clones. Nature, 355, 642–644. Pauly, T., Elbers, K., Ko¨ nig, M., Lengsfeld, T., Saalmu¨ller, A. and Thiel, H. J. (1995). Classical swine fever virus-specific cytotoxic T lymphocytes and identification of a T cell epitope. Journal of General Virology, 76, 3039–3049. Pauly, T., Ko¨nig, M., Thiel, H. J. and Saalmu¨ller, A. (1998). Infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine T lymphocytes. Journal of General Virology, 79, 31–40. Roitt, I., Brostoff, J. and Male, D. (1998). Receptores de las ce´lulas T y mole´culas del CPH. In: Inmunologı´a, 4th Edit., I. Roitt, J. Brostoff and D. Male, Eds, Harcourt Brace, Madrid, pp. 5.1–5.9. Ru¨menapf, T., Stark, R., Meyers, G. and Thiel, H. J. (1991). Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. Journal of Virology, 65, 589–597. Ryan, E. J., McNeela, E., Pizza, M., Rappuoli, R., O’Neil, L. and Mill, K. H. G. (2000). Modulation of innate and acquired immune response by Escherichia coli heat labile toxin: distinct pro- and anti-inflammatory effects of the non-toxic AB complex and the enzyme activity. Journal of Immunology, 165, 5750–5759. Samuel, C. E. (1991). Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology, 183, 1–11. Samuel, C. E. (1998). Reoviruses and the interferon system. Current Topics in Microbiology and Immunology, 233, 125–145. Samuel, C. E. (2001). Antiviral action of interferons. Clinical Microbiology Reviews, 14, 778–809. Sa´nchez-Cordo´n, P. J., Romanini, S., Salguero, F. J., Nu´n ˜ ez, A., Bautista, M. J., Jover, A. and Go´mezVillamandos, J. C. (2002). Apoptosis of thymocytes related to cytokine expression in experimental classical swine fever. Journal of Comparative Pathology, 127, 239–248. Sa´nchez-Cordo´n, P. J., Romanini, S., Salguero, F. J., RuizVillamor, E., Carrasco, L. and Go´mez-Villamandos, J. C. (2003). A histopathological, immunohistochemical and ultrastructural study of the intestine in pigs inoculated with classical swine fever virus. Veterinary Pathology, 40, 254–262.
260
P. J. Sa´nchez-Cordo´n et al.
Sato, M., Mikami, O., Kobayashi, M. and Nakajima, Y. (2000). Apoptosis in the lymphatic organs of piglets inoculated with classical swine fever virus. Veterinary Microbiology, 75, 1–9. Stasny, B. R., de Guise, S., Rompato, G. and Garmendia, A. E. (2001). Functional characterization of a swine CD4C/CD8C double positive lymphoblastoid T-cell line with a CD25C/CD45RAK phenotype generated in vitro with IL-2. Veterinary Immunology and Immunopathology, 78, 57–70. Straus, D. B. and Weiss, A. (1993). The CD3 chains of the T cell antigen receptor associated with the ZAP-70 tyrosine kinase are tyrosine phosphorylated after receptor stimulation. Journal of Experimental Medicine, 178, 1523–1530. Summerfield, A., Kno¨tig, S. M. and McCullough, K. C. (1998). Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. Journal of Virology, 72, 1853–1861. Summerfield, A., McNeilly, F., Walker, I., Allan, G., Knoeting, S. M. and McCullough, K. C. (2001). Depletion of CD4C and CD8highC T-cells before the onset of viraemia during classical swine fever. Veterinary Immunology and Immunopathology, 78, 3–19. Summerfield, A., Rziha, H. J. and Saalmu¨ller, A. (1996). Functional characterization of porcine CD4CCD8C extrathymic T lymphocytes. Cellular Immunology, 168, 291–296. Suradhat, S., Intrakamhaeng, M. and Damrongwatanapokin, S. (2001). The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection. Veterinary Immunology and Immunopathology, 83, 177–189.
Tanaka, N., Sato, M., Lamphier, M., Nozawa, H., Oda, E., Noguchi, S., Schreiber, R. D., Tsujimoto, Y. and Taniguchi, T. (1998). Type 1 interferons are essential mediators of apoptotic death in virally infected cells. Genes Cells, 3, 29–37. Tizard, I. R. (1998). Ce´lulas T auxiliaries y su respuesta al antı´geno. In: Inmunologı´a Veterinaria, 5th Edit., I. R. Tizard, Ed., McGraw-Hill Interamericana Editores, Me´xico, pp. 119–131. Van Miert, A. (1995). Pro-inflammatory cytokines in a ruminant model: pathophysiological, pharmacological, and therapeutic aspects. Veterinary Quarterly, 175, 41–50. Van Reeth, K., Nauwynck, H. and Pensaert, M. (1998). Production of interferon alpha, tumor necrosis factor alpha, interleukin-1 and inflammation during acute influenza in pigs: a possible model for humans? Journal of Infectious Diseases, 177, 1076–1079. Van Snick, J. (1990). Interleukin-6: an overview. Annual Review of Immunology, 8, 253–278. Wensvoort, G., Terpstra, C., de Kluijver, E. P., Kragten, C. and Warnaar, J. C. (1989). Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Veterinary Microbiology, 21, 9–20. Wood, P. R. and Seow, H. F. (1996). T cell cytokines and disease prevention. Veterinary Immunology and Immunopathology, 54, 33–34.
Received; June 7th; 2004 Accepted; October 18th; 2004