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Antigen presenting cells and B-cells in the pig
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Veterinary immunology
and
Veterinary Immunology and Immunopathology 43 (1994) 63-68
immunopatholo~y
Antigen presenting cells and B-cells in the pig A.T.J. Bianchi*, P.J. van der H e i j d e n Department of Immunology, DLO-Central Veterinary Institute, P.O. Box 65, Lelystad, Netherlands
Abstract
The central position of antigen presenting cells (APC) in the immune system and the heterogeneity of the APC family are discussed; both aspects are illustrated with data from species other than the pig. Thereafter the limited work on porcine APC is reviewed. The section on B-cells, the effector cells of the humoral immune system, exclusively focuses on 'porcine data'.
1. Antigen presenting cells
Many cell types are able to present antigen to the immune system, but the 'professional' cell types involved are macrophages, dendrocytes and B-cells (Leenen and Campbell, 1993 ). Antigen presenting cells (APC) present antigen associated with major histocompatibility complex (MHC) class I or II molecules to T-cells and provide accessory cell functions to T-cells (Steinman and Young, 1991 ). Antigen presentation to T-cells can result in activation or suppression of humoral or cellular effector mechanisms of the immune system. The type of induced effector mechanism depends on the type of T-helper (Th) cell that is activated by an APC. It is well documented in mice (Mosmann and Coffman, 1989 ) that at least two types of Th cells can be distinguished: activated Th 1 cells secrete cytokines such as 7IFN and IL2, mediate DTH like reactions and facilitate e.g. cytotoxic T-cell activity and IgG2a responses. Activated Th2 cells secrete cytokines such as IL4 and IL5 and facilitate antibody responses, e.g. IgE and IgA responses. These two types of Th cells also appear to be mutually inhibitory. This form of immune regulation by different types of Th cells has also been demonstrated for humans (Kapsenberg et al., 1992) and, most probably, comparable subsets of regulatory Th cell subsets exist in the pig. *Corresponding author. 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0165-2427 ( 94 ) 06009-0
64
A.T.J. Bianchi, P.J. van der Heijden / Vet. Immunol. lmmunopathol. 43 (1994) 63-68
The central issue is: what determines which type of Th cell becomes activated by an antigen? Although the antigen type and the route by which the antigen enters the host are important factors, the APC type involved in antigen presentation will finally determine the type of Th cell that becomes activated and thereby the type of immune response induced (Daynes et al., 1990). The major group of APC briefly discussed here consists of the macrophages and dendrocytes that belong to the so called mononuclear phagocyte system (Van Furth et al., 1972). One of the main characteristics of the mononuclear phagocytes is their large heterogeneity (Leenen and Campbell, 1993). This heterogeneity is based on their differentiation but also on their micro environment and functional capacities (Gordon et al., 1988 ). So, dendrocytes or interdigitating dendritic cells, mainly localised in the T-cell areas of lymphoid organs, are important for induction of primary immune responses (Steinman and Young, 1991 ). Classic tissue macrophages however, are probably involved in memory T-cell responses (Leenen and Campbell, 1993 ), whereas alveolar macrophages are mainly involved in immunosuppression (Thepen et al., 1989). The analysis of the in vivo APC function of mononuclear phagocytes requires in situ characterization of the various mononuclear phagocyte types. For a long time, this characterization was based on criteria such as morphology, presence of CR3 and Fc receptors, and histochemical staining of lysosomal enzymes (acid phosphatase and esterase staining). These criteria have also been used for porcine studies on mononuclear phagocytes (reviewed by Binns, 1982; Ramos et al., 1992), but are not selective for mononuclear phagocytes. A major improvement for in situ study of mononuclear phagocytes was achieved when large panels of monoclonal antibodies (mAb) against markers on mononuclear phagocytes became available for species such as human, mouse (Leenen and Campbell, 1993 ) and rat (Dijkstra et al., 1985 ). Some mAb are selective for certain mononuclear phagocytes, but there is no mAb that selectively detects all mononuclear phagocytes in all tissues. Therefore, combinations of different mAb are used for in situ characterization and functional study of mononuclear phagocytes. The work of Dijkstra and coworkers (1985) on rat mononuclear phagocytes illustrates the possibility of characterizing most mononuclear phagocytes in the body with a selected panel of just three mAb. For the pig, no mAb are available that react exclusively with mononuclear phagocytes; some of the mAb reviewed by Lunney and Pescovitz ( 1988 ) bind to subpopulations of mononuclear phagocytes and other cell types. During the first Swine CD workshop in 1992 these mAb were assigned to the following clusters: CD 1 (on B-cells and Langerhans cells, Pescovitz et al., 1990), SWC 1 (on T-cells and macrophages) and SWC3a ("the only real myeloid marker for the pig", on macrophages and granulocytes; Lunney, 1993 ). Saalmiiller and Reddehase ( 1988 ) demonstrated that monocytes could be sorted out from peripheral blood cells (PBL) after double staining for the SWC1 and SWC3a marker by flow cytometry. In addition to these three determinants the mAb against porcine MHC class II molecules (Lunney and Pescovitz, 1988) are useful in studying dendrocytes and activated macrophages (Leenen and Campbell, 1993). Other cluster determinants from the Swine CD Workshop which may be relevant for studying por-
A.T.J. Bianchi, P.J. van der Heijden / Vet. Immunol. ImmunopathoL 43 (1994) 63-68
65
cine mononuclear phagocytes are CD 18a (LFA 1 fl chain), CD21 (on B-cells and follicular dendritic cells) and CD25 (low affinity IL-2 receptor; Bailey et al., 1992). Pol and co-workers ( 1987 ) claimed a mAb (CVI-SwNL517.2 ) that specifically distinguished porcine alveolar macrophages and tissue macrophages, but this mAb was not submitted for the Swine CD workshop. Mononuclear phagocytes not only have a central position in the regulation of the immune response but they are often one of the main target cells of various pathogens that can severely hamper mononuclear phagocyte functions. Examples of such pathogens in pigs are African swine fever virus (Gonzales et al., 1992 ), pseudorabies virus (Chinsakchai and Mollitor, 1992), and Lelystad virus (Wensvoort et al., 1991 ). It is clear that research on these economically important diseases justifies research for basic knowledge of APC localisation and function in the pig, which is lacking. The development of mAb that selectively identify porcine mononuclear phagocytes will be a prerequisite for basic research.
2. B cells
2.1. Reagents and techniques to study porcine B cells The study of the porcine B-cell compartment started more than 20 years ago when porcine Ig-isotypes were isolated and polyclonal antisera specific for porcine IgM, IgG and IgA developed (Curtis and Bourne, 1971; Allen and Porter, 1973). These antisera have been used for studying the porcine B-cells or their secreted immunoglobulins by: (i) in situ localisation of B-cells (surface immunoglobulin positive cells) and plasma cells (immunoglobulin containing cells) by immunohistology (Allen and Porter, 1973; Brown and Bourne, 1976 ); (ii) quantitative analysis of immunoglobulin isotypes in porcine serum or excreta by radial immunodiffusion (Curtis and Bourne, 1971; Butler et al., 1981 ); (iii) quantification of antibody secreting cells or immunoglobulin secreting cells in porcine lymphoid cell suspensions with a plaque forming cell assay (PFC-assay) (Bushmann et al., 1974; Scheffel et al., 1979 ) or a reversed-PFC assay (Scheffel et al., 1981 ) respectively. More recently mAb against porcine immunoglobulin isotypes and subclasses (Van Zaane and Hulst, 1987; Lunney and Pescovitz, 1988 ) have been developed which provided a definite solution for specificity problems and batch to batch variation which are often the major problems of assays based on polyclonal antisera. These isotype-specific mAb are also used for studying the B-cell compartment by the three approaches listed above. Although mAb are not appropriate for radial immunodiffusion or PFC assays, they can easily be applied in enzymelinked immunosorbent assays (ELISAs) to quantify porcine Ig-isotypes (Bianchi and Van der Heijden, 1993 ) and in antigen-specific ELIspot assays (Russell et al., 1987; Bianchi et al., 1990; VanCott et al., 1993) or reversed ELIspot assays (Bianchi et al., 1991 ) to quantify antibody- or immunoglobulin-secreting cells respectively.
66
A.T.J. Bianchi, P.,L van der Heijden / Vet. lmmunol. Immunopathol. 43 (1994) 63-68
The reagents and techniques described here are mostly used for in vivo studies of the porcine B-cell compartment, although porcine B-cell responses have also been studied in vitro after stimulation by antigen (Hammerberg and Schurig, 1984) or by polyclonal stimulators such as pokeweed mitogen (PWM) (Scheffel et al., 1981 ) and lipopolysaccharide (LPS) (Symons and Lay, 1978 ). 2.2. Research on porcine B cells
The development of immune responses in early pig ontogeny and the porcine immunoglobulins are reviewed in detail in separate papers of this issue (Butler and Brown, 1994; Tlaskalova et al., 1994). Therefore, the last part of this section is confined to: (i) the distribution of B-cell subsets in various (lymphoid) tissues of young pigs and (ii) the arguments regarding the function of porcine ileal Peyer's patch as a 'primary B-cell organ'. B-cells in mucosal tissues Most studies on the distribution of porcine B-cells are focused on B-cells in mucosal tissues. This reflects the veterinary interest of these studies knowing that most infectious diseases have their porte d'entrre via mucosal surfaces. During the first period of life the intestinal IgM-containing cells outnumber the IgA-containing cells; thereafter IgA becomes the predominant isotype (Allen and Porter, 1973; Brown and Bourne, 1976; Butler et al., 1981; Bianchi et al., 1992). Most Ig-containing cells are located in the crypt region of the porcine intestine. It is clear for the pig, as for humans (Brandtzaeg et al., 1985 ), that IgM and IgA are both important for intestinal B-cell responses. In this respect humans and pigs clearly differ from mice which do not show any IgM-containing cells along the intestinal mucosa at any age (Van der Heijden et al., 1987). The situation is different along the porcine upper respiratory tract, because IgA-containing cells predominates in this mucosal tissue already in the first period of life (Bradley et al., 1976 ). This is not reflected by a predominance of IgA in nasal secretions, since in these secretions higher IgG concentrations were described (Morgan and Bourne, 1981 ). Unlike humans (Brandtzaeg et al., 1985 ), no data are available regarding IgA subclass distribution in porcine (mucosal) tissues. However, such data can be expected in the near future. The existence of two porcine IgA subclasses is indicated by the observations that two different IgA specific mAb show a complementary staining pattern in immunohistology and a complementary detection of IgA-secreting cells in the reversed ELIspot (A.T.J. Bianchi et al., unpublished observation, 1993 ). However, this is not compatible with recent data of Butler and Brown (1994) that indicate the existence of one porcine ot gene. B-cells in systemic lymphoid organs The data on porcine B-cell subsets in systemic lymphoid organs are very limited. A study by Bianchi and co-workers (1992) is the only one that gives a complete picture of the isotype distribution of Ig-containing cells over the systemic
A.T.J. Bianchi, P.J. van der Heijden / Vet. lmmunol, lmmunopathol. 43 (1994) 63-68
67
lymphoid organs. A remarkable observation from their study is the low incidence of IgG-containing cells in the systemic lymphoid organs especially in the spleen, whereas IgG is still the far most predominant isotype in porcine serum (Brown and Bourne, 1976; Butler et al., 1981 ). Thus, in pigs, the Ig-containing cells isotypes of the various systemic lymphoid organs together do not correlate with the Ig-isotype concentration in serum. Positive correlations have been observed for the various Ig-isotypes in humans but not in mice (Benner et al., 1982).
The function of the porcine ileal Peyer's patch Binns and Licence (1985) provided evidence for a functional difference between the jejunal Peyer's patches (j-PP) and ileal Peyer's patch (i-PP) of the pig. They observed less lymphocyte migration into the i-PP than into the j-PP. The iPP also had smaller interfollicular areas compared with the j-PP and therefore a higher incidence of B cells. These results suggest that porcine i-PP may have a function as primary (B-cell) lymphoid organ as has been described in lambs (Reynolds, 1987a). The difference between porcine j-PP and i-PP was further substantiated by Pabst and co-workers ( 1988 ) who demonstrated that the follicles of i-PP not only increased in size but also increased in number. However, the rate of lymphocyte production in the follicles increased dramatically with age in both types of porcine PP (Pabst et al., 1988), whereas in lambs the production rate ofi-PP is independent of antigen and microbial contact (Reynolds, 1987b). Apoptosis causing extensive B-cell death was demonstrated in the ovine i-PP as in chicken bursa and is associated with the function as primary lymphoid organ (Motyka and Reynolds, 1991 ). Such data are not available for the pig. Lymphoid follicles in porcine PP are also found before birth at 50 days of gestation (Chapman et al., 1974), but more recent studies clearly demonstrated that further development after birth in both types of porcine PP was strongly dependent on antigenic stimulation (Rothk~Stter and Pabst, 1989; Barman et al., 1994). Nevertheless, the time course of the development of both types of porcine PP differ (Barman et al., 1994). Moreover, the i-PP regresses first in pigs (Pabst et al., 1988) as in lambs. Taken together these data lead to the conclusion that the porcine i-PP does not have all the characteristics of a primary lymphoid organ like the ovine i-PP, but it is obvious that there is a functional difference between the j-PP and i-PP of pigs. One can speculate about a role of porcine i-PP in induction of (mucosal) immunity or oral tolerance especially during the first period of life.
References W.D. Allen and P. Porter (1973), Immunology 24, 493. M. Bailey, K. Stevens, P.W. Bland and C.R. Stokes ( 1992 ), J. Immunol. Meth. 153, 85. N.N. Barman, H.J. Rothk6tter, A.T.J. Bianchi, J.J. Zwart and R. Pabst, submitted. R. Benner et al. (1982), Immunol. Today, 3, 243. A.T.J. Bianchi and P.J. van der Heijden (1993), Monogr. Vet. Immunol. 2.
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A.T.J. Bianchi, J.W. Scholten, I.M.C.H. Jongenelen and G. Koch (1990), Vet. Immunol. Immunopathol. 24, 125. A.T.J. Bianchi, B. Moonen, J.W. Scholten and P.J. van der Heijden (1991), in Lymphatic Tissues and in vivo Immune Responses, B.A. Imhof, S. Berrih-Aknin and S. Ezine, Eds. (Marcel Dekker, New York, p. 407. A.T.J. Bianchi, R.J. Zwart, S.H.M. Jeurissen and H.W.M. Moonen-Leusen (1992), Vet. Immunol. Immunopathol. 33, 201. R.M. Binns (1982), Vet. Immunol. Immunopathol. 3, 95. R.M. Binns and S.T. Licence (1985), Adv. Exp. Med. Biol. 186, 661. P.A. Bradley, F.J. Bourne and P.J. Brown ( 1976 ), Vet. Pathoh 13, 8 I. P. Brandtzaeg et al. ( 1985 ), Scan. J. Gastroenterol. Suppl. 17. P.J. Brown and F.J. Bourne (1976), Am. J. Vet. Res. 37, 9. H. Buschmann, V. Junge, H. Krausslich and A. Radzikowski (1974), Med. Microbiol. Immunol. 159, 179. J.E. Butler and W.R. Brown (1994), Vet. Immunol. Immunopathol (this issue). J.E. Butler, F. Klobasa and E. Werhahn ( 1981 ), Vet. Immunol. Immunopathol. 2, 53. H.A. Chapman, J.S. Johnson and M.D. Cooper (1974), J. Immunol. 112,555. S. Chinsakchai and T.W. Molitor (1992), Vet. Immunol. Immunopathol. 30, 247. J. Curtis and F.J. Bourne ( 1971 ), Biochim. Biophys. Acta 236, 319. R.A. Daynes, B.A. Araneo, T.A. Dowell, K. Huang and R. Dudley (1990), J. Exp. Med. 17 l, 979. C.D. Dijkstra, E.A. D6pp, P. Joling and G. Kraal (1985), Immunology 54, 589. M. Gonzalez Juarrero et al. ( 1992 ), Vet. Immunoh Immunopathol. 32, 243. S. Gordon, S. Keshav and L.P. Chung (1988), Curr. Opin. Immunol. 1, 26. C. Hammerberg and G.G. Schurig (1984), Vet, lmmunol. Immunopathol. 7, 139. M.L. Kapsenberg, H.M. Jansen, J.D. Bos and E.A. Wierenga ( 1992 ), Curr. Opin. Immunol. 4, 788. P.J.M. Leenen and P.A. Campbell (1993), in Blood Cell Biochemistry, Vol. V, Macrophages and Related Cells, M.A. Horton, Ed. (Plenum Press, New York), p. 29. J.K. Lunney ( 1993 ), Immunol. Today 14, 147. J.K. Lunney and M.D. Pescovitz (1988), in Differentiation Antigens in Lymphohaemopoietic Tissues, M. Miyasaka and Z. Trnka, Eds. (Marcel Dekker, New York), Immunology Series, Vol. V, p. 38. K.L. Morgan and F.J. Bourne ( 1981 ), Res. Vet. Sci. 31,40. T.R. Mosmann and R.L. Coffman (1989), Annu. Rev. Immunol. 7, 145. B. Motyka and J.D. Reynolds ( 1991 ), Eur. J. Immunot. 21, 1951. R. Pabst, M. Geist, H.J. Rothk6tter and F.J. Fritz (1988), Immunology 64, 539. M.D. Pescovitz et al. (1990), Tissue antigens 35, 151. J.M.A. Pol et al. (1987), Abstract of a Symposium on African Swine Fever and Pig Immunology, Salamanca, Spain, p. 48 (Unpublished.) J.A. Ramos et al. (1992), Am. J. Vet. Res. 53, 1418. J.P. Reynolds (1987a), Curr. Top. Microbiol. Immunol. 135, 43. J.P. Reynolds (1987b), Eur. J. Immunol. 17, 503. H.J. Rothk/Stter and R. Pabst ( 1989 ), Immunology 67, 103, P.H. Russell, D.K.J. Mackay, I. Odzemir and N.M. Mackenzie (1987), J. Immunol. Methods 101, 229. A. Saalmtiller and M.J. Reddehase ( 1988 ), Res. Vet. Sci. 45, 311. J.W. Scheffel, T.M. Setcavage and Y.B. Kim (1979), Cell. Immunol. 44, 109. J.W. Scheffel, T.M. Setcavage and Y.B. Kim ( 1981 ), Dev. Comp. Immunol. 5, 313. R.M. Steinman and J.W. Young ( 1991 ), Curr. Opin. Immunol. 3, 361. D.B.A. Symons and C.A. Lay ( 1978 ), Int. Arch. Allergy Appl. Immunol. 57, 418. H. Tlaskalova-Hogenova, I. Trehichavsky, F. Kovaru and J, Sterzl (1994), Vet. Immunol. Immunopathol. 43 (this issue). T. Thepen, N. van Rooyen and G. Kraal ( 1989 ), J. Exp. Med. 170, 499. J.L. VanCott, T.A. Brim, R.A. Simkins and L.J. Sail ( 1993 ), J. Immunol. 150, 3990. P.J. van der Heijden, W. Stok and A.T.J. Bianchi ( 1987 ), Immunology 62, 551. R. van Furth et al. (1972), Bull. WHO 46, 845. D. van Zaane and M.M. Hulst (1987), Vet. lmmunol. Immunpathol. 16, 23. G. Wensvoort et al. ( 1991 ), Vet. Q. 13, 121.
Veterinary immunology
and
Veterinary Immunology and Immunopathology 43 (1994) 63-68
immunopatholo~y
Antigen presenting cells and B-cells in the pig A.T.J. Bianchi*, P.J. van der H e i j d e n Department of Immunology, DLO-Central Veterinary Institute, P.O. Box 65, Lelystad, Netherlands
Abstract
The central position of antigen presenting cells (APC) in the immune system and the heterogeneity of the APC family are discussed; both aspects are illustrated with data from species other than the pig. Thereafter the limited work on porcine APC is reviewed. The section on B-cells, the effector cells of the humoral immune system, exclusively focuses on 'porcine data'.
1. Antigen presenting cells
Many cell types are able to present antigen to the immune system, but the 'professional' cell types involved are macrophages, dendrocytes and B-cells (Leenen and Campbell, 1993 ). Antigen presenting cells (APC) present antigen associated with major histocompatibility complex (MHC) class I or II molecules to T-cells and provide accessory cell functions to T-cells (Steinman and Young, 1991 ). Antigen presentation to T-cells can result in activation or suppression of humoral or cellular effector mechanisms of the immune system. The type of induced effector mechanism depends on the type of T-helper (Th) cell that is activated by an APC. It is well documented in mice (Mosmann and Coffman, 1989 ) that at least two types of Th cells can be distinguished: activated Th 1 cells secrete cytokines such as 7IFN and IL2, mediate DTH like reactions and facilitate e.g. cytotoxic T-cell activity and IgG2a responses. Activated Th2 cells secrete cytokines such as IL4 and IL5 and facilitate antibody responses, e.g. IgE and IgA responses. These two types of Th cells also appear to be mutually inhibitory. This form of immune regulation by different types of Th cells has also been demonstrated for humans (Kapsenberg et al., 1992) and, most probably, comparable subsets of regulatory Th cell subsets exist in the pig. *Corresponding author. 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0165-2427 ( 94 ) 06009-0
64
A.T.J. Bianchi, P.J. van der Heijden / Vet. Immunol. lmmunopathol. 43 (1994) 63-68
The central issue is: what determines which type of Th cell becomes activated by an antigen? Although the antigen type and the route by which the antigen enters the host are important factors, the APC type involved in antigen presentation will finally determine the type of Th cell that becomes activated and thereby the type of immune response induced (Daynes et al., 1990). The major group of APC briefly discussed here consists of the macrophages and dendrocytes that belong to the so called mononuclear phagocyte system (Van Furth et al., 1972). One of the main characteristics of the mononuclear phagocytes is their large heterogeneity (Leenen and Campbell, 1993). This heterogeneity is based on their differentiation but also on their micro environment and functional capacities (Gordon et al., 1988 ). So, dendrocytes or interdigitating dendritic cells, mainly localised in the T-cell areas of lymphoid organs, are important for induction of primary immune responses (Steinman and Young, 1991 ). Classic tissue macrophages however, are probably involved in memory T-cell responses (Leenen and Campbell, 1993 ), whereas alveolar macrophages are mainly involved in immunosuppression (Thepen et al., 1989). The analysis of the in vivo APC function of mononuclear phagocytes requires in situ characterization of the various mononuclear phagocyte types. For a long time, this characterization was based on criteria such as morphology, presence of CR3 and Fc receptors, and histochemical staining of lysosomal enzymes (acid phosphatase and esterase staining). These criteria have also been used for porcine studies on mononuclear phagocytes (reviewed by Binns, 1982; Ramos et al., 1992), but are not selective for mononuclear phagocytes. A major improvement for in situ study of mononuclear phagocytes was achieved when large panels of monoclonal antibodies (mAb) against markers on mononuclear phagocytes became available for species such as human, mouse (Leenen and Campbell, 1993 ) and rat (Dijkstra et al., 1985 ). Some mAb are selective for certain mononuclear phagocytes, but there is no mAb that selectively detects all mononuclear phagocytes in all tissues. Therefore, combinations of different mAb are used for in situ characterization and functional study of mononuclear phagocytes. The work of Dijkstra and coworkers (1985) on rat mononuclear phagocytes illustrates the possibility of characterizing most mononuclear phagocytes in the body with a selected panel of just three mAb. For the pig, no mAb are available that react exclusively with mononuclear phagocytes; some of the mAb reviewed by Lunney and Pescovitz ( 1988 ) bind to subpopulations of mononuclear phagocytes and other cell types. During the first Swine CD workshop in 1992 these mAb were assigned to the following clusters: CD 1 (on B-cells and Langerhans cells, Pescovitz et al., 1990), SWC 1 (on T-cells and macrophages) and SWC3a ("the only real myeloid marker for the pig", on macrophages and granulocytes; Lunney, 1993 ). Saalmiiller and Reddehase ( 1988 ) demonstrated that monocytes could be sorted out from peripheral blood cells (PBL) after double staining for the SWC1 and SWC3a marker by flow cytometry. In addition to these three determinants the mAb against porcine MHC class II molecules (Lunney and Pescovitz, 1988) are useful in studying dendrocytes and activated macrophages (Leenen and Campbell, 1993). Other cluster determinants from the Swine CD Workshop which may be relevant for studying por-
A.T.J. Bianchi, P.J. van der Heijden / Vet. Immunol. ImmunopathoL 43 (1994) 63-68
65
cine mononuclear phagocytes are CD 18a (LFA 1 fl chain), CD21 (on B-cells and follicular dendritic cells) and CD25 (low affinity IL-2 receptor; Bailey et al., 1992). Pol and co-workers ( 1987 ) claimed a mAb (CVI-SwNL517.2 ) that specifically distinguished porcine alveolar macrophages and tissue macrophages, but this mAb was not submitted for the Swine CD workshop. Mononuclear phagocytes not only have a central position in the regulation of the immune response but they are often one of the main target cells of various pathogens that can severely hamper mononuclear phagocyte functions. Examples of such pathogens in pigs are African swine fever virus (Gonzales et al., 1992 ), pseudorabies virus (Chinsakchai and Mollitor, 1992), and Lelystad virus (Wensvoort et al., 1991 ). It is clear that research on these economically important diseases justifies research for basic knowledge of APC localisation and function in the pig, which is lacking. The development of mAb that selectively identify porcine mononuclear phagocytes will be a prerequisite for basic research.
2. B cells
2.1. Reagents and techniques to study porcine B cells The study of the porcine B-cell compartment started more than 20 years ago when porcine Ig-isotypes were isolated and polyclonal antisera specific for porcine IgM, IgG and IgA developed (Curtis and Bourne, 1971; Allen and Porter, 1973). These antisera have been used for studying the porcine B-cells or their secreted immunoglobulins by: (i) in situ localisation of B-cells (surface immunoglobulin positive cells) and plasma cells (immunoglobulin containing cells) by immunohistology (Allen and Porter, 1973; Brown and Bourne, 1976 ); (ii) quantitative analysis of immunoglobulin isotypes in porcine serum or excreta by radial immunodiffusion (Curtis and Bourne, 1971; Butler et al., 1981 ); (iii) quantification of antibody secreting cells or immunoglobulin secreting cells in porcine lymphoid cell suspensions with a plaque forming cell assay (PFC-assay) (Bushmann et al., 1974; Scheffel et al., 1979 ) or a reversed-PFC assay (Scheffel et al., 1981 ) respectively. More recently mAb against porcine immunoglobulin isotypes and subclasses (Van Zaane and Hulst, 1987; Lunney and Pescovitz, 1988 ) have been developed which provided a definite solution for specificity problems and batch to batch variation which are often the major problems of assays based on polyclonal antisera. These isotype-specific mAb are also used for studying the B-cell compartment by the three approaches listed above. Although mAb are not appropriate for radial immunodiffusion or PFC assays, they can easily be applied in enzymelinked immunosorbent assays (ELISAs) to quantify porcine Ig-isotypes (Bianchi and Van der Heijden, 1993 ) and in antigen-specific ELIspot assays (Russell et al., 1987; Bianchi et al., 1990; VanCott et al., 1993) or reversed ELIspot assays (Bianchi et al., 1991 ) to quantify antibody- or immunoglobulin-secreting cells respectively.
66
A.T.J. Bianchi, P.,L van der Heijden / Vet. lmmunol. Immunopathol. 43 (1994) 63-68
The reagents and techniques described here are mostly used for in vivo studies of the porcine B-cell compartment, although porcine B-cell responses have also been studied in vitro after stimulation by antigen (Hammerberg and Schurig, 1984) or by polyclonal stimulators such as pokeweed mitogen (PWM) (Scheffel et al., 1981 ) and lipopolysaccharide (LPS) (Symons and Lay, 1978 ). 2.2. Research on porcine B cells
The development of immune responses in early pig ontogeny and the porcine immunoglobulins are reviewed in detail in separate papers of this issue (Butler and Brown, 1994; Tlaskalova et al., 1994). Therefore, the last part of this section is confined to: (i) the distribution of B-cell subsets in various (lymphoid) tissues of young pigs and (ii) the arguments regarding the function of porcine ileal Peyer's patch as a 'primary B-cell organ'. B-cells in mucosal tissues Most studies on the distribution of porcine B-cells are focused on B-cells in mucosal tissues. This reflects the veterinary interest of these studies knowing that most infectious diseases have their porte d'entrre via mucosal surfaces. During the first period of life the intestinal IgM-containing cells outnumber the IgA-containing cells; thereafter IgA becomes the predominant isotype (Allen and Porter, 1973; Brown and Bourne, 1976; Butler et al., 1981; Bianchi et al., 1992). Most Ig-containing cells are located in the crypt region of the porcine intestine. It is clear for the pig, as for humans (Brandtzaeg et al., 1985 ), that IgM and IgA are both important for intestinal B-cell responses. In this respect humans and pigs clearly differ from mice which do not show any IgM-containing cells along the intestinal mucosa at any age (Van der Heijden et al., 1987). The situation is different along the porcine upper respiratory tract, because IgA-containing cells predominates in this mucosal tissue already in the first period of life (Bradley et al., 1976 ). This is not reflected by a predominance of IgA in nasal secretions, since in these secretions higher IgG concentrations were described (Morgan and Bourne, 1981 ). Unlike humans (Brandtzaeg et al., 1985 ), no data are available regarding IgA subclass distribution in porcine (mucosal) tissues. However, such data can be expected in the near future. The existence of two porcine IgA subclasses is indicated by the observations that two different IgA specific mAb show a complementary staining pattern in immunohistology and a complementary detection of IgA-secreting cells in the reversed ELIspot (A.T.J. Bianchi et al., unpublished observation, 1993 ). However, this is not compatible with recent data of Butler and Brown (1994) that indicate the existence of one porcine ot gene. B-cells in systemic lymphoid organs The data on porcine B-cell subsets in systemic lymphoid organs are very limited. A study by Bianchi and co-workers (1992) is the only one that gives a complete picture of the isotype distribution of Ig-containing cells over the systemic
A.T.J. Bianchi, P.J. van der Heijden / Vet. lmmunol, lmmunopathol. 43 (1994) 63-68
67
lymphoid organs. A remarkable observation from their study is the low incidence of IgG-containing cells in the systemic lymphoid organs especially in the spleen, whereas IgG is still the far most predominant isotype in porcine serum (Brown and Bourne, 1976; Butler et al., 1981 ). Thus, in pigs, the Ig-containing cells isotypes of the various systemic lymphoid organs together do not correlate with the Ig-isotype concentration in serum. Positive correlations have been observed for the various Ig-isotypes in humans but not in mice (Benner et al., 1982).
The function of the porcine ileal Peyer's patch Binns and Licence (1985) provided evidence for a functional difference between the jejunal Peyer's patches (j-PP) and ileal Peyer's patch (i-PP) of the pig. They observed less lymphocyte migration into the i-PP than into the j-PP. The iPP also had smaller interfollicular areas compared with the j-PP and therefore a higher incidence of B cells. These results suggest that porcine i-PP may have a function as primary (B-cell) lymphoid organ as has been described in lambs (Reynolds, 1987a). The difference between porcine j-PP and i-PP was further substantiated by Pabst and co-workers ( 1988 ) who demonstrated that the follicles of i-PP not only increased in size but also increased in number. However, the rate of lymphocyte production in the follicles increased dramatically with age in both types of porcine PP (Pabst et al., 1988), whereas in lambs the production rate ofi-PP is independent of antigen and microbial contact (Reynolds, 1987b). Apoptosis causing extensive B-cell death was demonstrated in the ovine i-PP as in chicken bursa and is associated with the function as primary lymphoid organ (Motyka and Reynolds, 1991 ). Such data are not available for the pig. Lymphoid follicles in porcine PP are also found before birth at 50 days of gestation (Chapman et al., 1974), but more recent studies clearly demonstrated that further development after birth in both types of porcine PP was strongly dependent on antigenic stimulation (Rothk~Stter and Pabst, 1989; Barman et al., 1994). Nevertheless, the time course of the development of both types of porcine PP differ (Barman et al., 1994). Moreover, the i-PP regresses first in pigs (Pabst et al., 1988) as in lambs. Taken together these data lead to the conclusion that the porcine i-PP does not have all the characteristics of a primary lymphoid organ like the ovine i-PP, but it is obvious that there is a functional difference between the j-PP and i-PP of pigs. One can speculate about a role of porcine i-PP in induction of (mucosal) immunity or oral tolerance especially during the first period of life.
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