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Effect of early weaning on the development of immune cells in the pig small intestine
Veterinary immunology
Veterinary Immunology and Immunopathology
and
immunopathology
44(1995)319-327
Effect of early weaning on the development of immune cells in the pig small intestine M.A. Vega-Lbpez”, M. Baileyb**, E. Telemob%*, CR. Stokesb ‘INIFAP-SARH, Mexico and FES-Cuautitlh-CM, Mexico bDivision of Molecular and Cellular Biology, Department of Clinical Veterinary Sciences, UniversityofBristol, Langford House, Langford, Bristol BS18 7011, UK
Accepted 9 February 1994
Abstract The controlled effects of age and weaning on the numbers of CD2 + T cells, subsets (CD4+, CD8+), accessory cells (macrophage/granulocyte) and cells expressing MHC class 11 (DQw ) and IL-2R in the piglet intestine was investigated. At birth low numbers of CD2+CD4_CD8cells were the only demonstrable T cells in the intestine. Monocyte/granulocyte and MHC class II+ cells were also detected in low numbers and IL-2R+ cells were proportionally quite numerous. All those cell populations, except the IL-2R+ cells, increased thereafter and peaked at Week 7 when the numbers of cells were comparable with those of adult animals. CD4+ cells increased dramatically after Week 1. In contrast, CD8+ remained scarce until after 5-7 weeks of age in unweaned animals. Four days after weaning at 3 weeks old, there were increases in CD2+ (PC 0.00 1) and macrophage/granulocyte (PC 0.0 1) cells in proximal small intestinal villi and in CD2+ cells only (PC 0.01) in crypts. No significant changes in cell numbers were demonstrated in the distal small intestine.
1. Introduction
At weaning, the young pig is abruptly subjected to a radical change in diet coupled with the waning of maternally derived serum immunoglobulins and the withdrawal of the locally protective elements of milk. The consequence of this * Corresponding author. ’ Present address: Clinical Immunology Department, University Hospital, Goteborg, 0165-2427/95/$09.50 0 1995 Elsevier Science B.V. AI1rights reserved SSDIOl65-2427(94)05309-G
Sweden.
320 M.A. Vega-L6pe.zet al. / Veterinary Immunology and Immunopathology 44 (1995) 319-327
may be to precipitate the onset of the syndrome of postweaning diarrhoea, characterised by villus atrophy, malabsorption due to enterocyte immaturity and bacterial overgrowth (Hampson, 1986; Hampson and Kidder, 1986). It has been postulated that an immune reaction (hypersensitivity) to fed protein may be involved in the early pathogenesis of this syndrome (Miller et al., 1983 ), predisposing to subsequent opportunistic infections (Escherichia co/i) which may prolong and exaggerate the situation (Miller et al., 1984). Because of the importance of enteric diseases to both animal and human health, the study of the immune system in the gut is crucial. We have recently reported a degree of organisation of the lymphoid cells within the diffuse lamina propria in mature pigs (VegaLopez et al., 1993). The appearance of such cells has been described by other workers in gnotobiotic piglets in low numbers after birth (Rothkotter et al., 199 1) and in piglets reared conventionally under systems involving weaning at 5 weeks of age (Bianchi et al., 1992). However, under the conditions of these experiments, the effects of weaning could not be separated from the effects of age and exposure to environment. In addition, postweaning disease is most severe in piglets weaned at 3 weeks of age. Here, we have examined changes in cells populating the intestine, in piglets maintained on the sow without access to solid food and in a controlled study after weaning. 2. Material and methods 2.1. Animals
All the. animals used were from a minimal disease herd of large white sows, maintained at the University of Bristol. Piglets suckling the sow were not allowed access to creep feed. Weaned piglets were fed a soya-based diet, the composition of which has been described (Miller et al., 1986). The animals were killed in accordance with local guidelines, with an overdose of sodium pentobarbitone (Euthatal, RMB Animal Health Ltd., Dagenham, UK). 2.2. Collection of samples
Samples from proximal ( 1 m from the pyloric junction) and distal ( 1 m from ileo-caecal junction) small intestine were either snap frozen in liquid nitrogen, or immediately fixed in paraformaldehyde-lysine-periodate fixative as described by Gendelman et al. ( 1983). Serial 2-4 pm sections of these tissues were prepared and used for immunohistochemical staining. 2.3. Monoclonal antibodies
Monoclonal antibodies recognising T cell markers and accessory cells were provided by Dr. J. Lunney, NIH Bethesda. The clones used were MSA4, anti-CD2 (Hammerberg and Schurig, 1986)) 74- 12-4, anti-CD4 and 76-2- 11, anti-CD8
M.A. Vega-L&e2 et al. / Veterinary Immunology and Immunopathology 44 (1995) 319-327
32 1
(Pescovitz et al., 1985) and 74-22-l 5, anti-monocyte/granulocyte (Hammerberg and Schurig, 1986). Clone H-42-A, recognising an MHC class II (DQw) determinant (Davis et al., 1987) was obtained from V.M.R.D., Washington. Clone 23 1.3B2, anti-IGZR, was generated in this laboratory (Bailey et al., 1992). 2.4. Immunohistochemistry The procedure used has been fully described by Vega-Mpez et al. ( 1993 ). Briefly, tissue sections were incubated with the optimal dilution of monoclonal antibody, then with an affinity purified, biotinylated rabbit anti-mouse antibody (Dakopatts, Glostrup, Denmark) followed by Streptavidin-horse radish peroxidase-avidin complex (Dakopatts). The reaction was visualised with 0.05% diaminobenzidine in 0.05 M Tris-HCl plus 0.01% hydrogen peroxide. The slides were counterstained with Harris haematoxylin and mounted in DPX. 2.5. Counts of cells Cells were counted using a high resolution, semi-automatic, computer assisted image analyser system (VIDS IV, Analytical Measuring Systems, Saffron Walden, UK). The positive cells were counted after the area in the lamina propria was calculated. The results were expressed as cells mm-*. At least five randomly selected fields in each tissue sample were counted, and because of the particular distribution of cells (Vega-Lopez et al,, 1993), the villus and crypt zones were counted separately. 2.4. Statistics The differences between groups were tested by analysis of variance using litter and treatment as blocks where appropriate. 3. Results 3. I. Changes in cell populations with age
Two litters of ten piglets each were selected and maintained on the sow without access to solid feed. Two piglets from each litter were killed at 0 (presuckled), 1, 3,5, and 7 weeks of age. The 6-month-old animals were described by Vega-Lopez et al. (1993). At birth, the few CD2+ cells were evenly distributed in the intestinal lamina propria. No staining for CD4/CD8 was apparent at this age and these cells were therefore CD4/CD8 negative (Fig. 1). Sites in the proximal and distal small intestine contained similar numbers of CD2+ cells. At all times after birth, these cells were more concentrated in villi than in crypts. The number of CD2+ cells in lamina propria and within the epithelium increased, reaching a maximum at
322 M.A. Vega-Lbpez et al. / Veterinary Immunology and Immunopathology 44 (1995) 319-327
Fig. 1. Intestine of newborn, presuckled piglet stained for CD2.
Weeks 5-7 (Fig. 2 (A) ). After the first week of age, an increase of CD4+ cells was also detectable within the lamina propria only. In contrast, CD8+ cells only moderately increased by the fifth week of age. The proportion of the CD2+ cells in the epithelial compartment increased from 38% at birth, to 53% at 7 weeks of age in the proximal small intestine, and up to 64% in the ileum. Low numbers of monocyte/granulocyte cells were present at birth with similar numbers of cells in the proximal small intestine and ileum (Fig. 2(B) ). These cells appeared to be evenly distributed in the lamina propria. After the first week of age, more cells were found in the distal than proximal small intestine. In addition the crypt zone was more populated than the villi. The number of cells reached a maximum at 5 weeks of age. IG2R+ cells were found in significant numbers at birth (Fig. 2 (C) ) but the numbers and distribution did not change significantly with age. These cells had large nuclei with prominent cytoplasm, and they were evenly distributed in the intestinal lamina propria. The numbers of MHC class II+ cells are shown in Fig. 2 (D). Even by 1 week of age, a dramatic increase in the number of positive cells was evident. The cells were concentrated in the villi, but significant numbers were also present in the crypts. Again, the density of cells peaked at Week 5 and this was most obvious in the villi. In the crypts, the number of positive cells remained quite stable with a transient drop in numbers at Week 3. Some of these cells showed dendritic morphology and were occasionally situated close to the epithelium. As previously reported, no staining for class II was detected in enterocytes.
en
2000
3000
4000
0
0
0
1
1
Weeks
3
3
7
7
6 months
6 months
\”
,”IA z 0
E 2
% E
2000
3000
4000
5000
0
0
1
WeeksofzkP
3
5
7
6 months
D
C
(A), the antigen recognised by the monoclonal antibody 74-22-l 5 (B), IL-2R (C) and MHC class II (D) piglets of increasing ages. Error bars are standard deviations of four animals.
of Age
5
5
Proljmal jetinal villi Pr‘,d,“al Jeju,d Crypts Distal jejunal wlli Distal jejunal crypts
Fig. 2. Mean numbers of cells expressingCD2 in intestinal lamina propria of litter-matched
c c-l
:: -z
E z
E
s
q
0
q q
2 z
324 M.A. Vega-Lbpez et al. I Veterinary Immunology and Immunopathology 44 (1995) 319-327
3.2. Eflect of weaning on cell populations
Two litters of ten piglets each were selected and at 2 1 days of age, live piglets from each litter were weaned onto a soya-containing diet. The remaining piglets were maintained on the sow without access to solid feed. All piglets were killed at 25 days of age. A comparison of weaned versus unweaned groups showed that in weaned animals there were significantly greater numbers of CD2+ (P
““““1T
E E iI? m 3
II
4000
T
1
CD2
MHC Class II
Cell surface
IL -2R
Mo/PMN
marker
Fig. 3. Mean numbers of cells expressing CD2, the antigen recognised by the monoclonal antibody 7422-15, MHC class II and IL-2R in villi (A) and cryps (B) of proximal jejunum of unweaned and 4 day weaned piglets. Error bars represent standard deviations of ten animals.
M.A. Vega-Lbpez et al. / Veterinary Immunology and Immunopathology
44 (1995) 319-327
325
4. Discussion The results observed in piglets of different ages are in agreement with those of other workers (Rothkotter et al., 199 1; Bianchi et al., 1992 ), but have been extended to include expression of the potentially modulatable antigens, MHC class II and interleukin 2 receptors. At birth, the majority of the T lymphocytes identified in the lamina propria of newborn animals lacked both the CD4 and CD8 markers. Many of these cells with unusual phenotype (CD2+CD4_CD8) were located in the epithelium. This dual-negative population of cells has been described in adult pig blood (Saalmuller et al., 1989), and recently in pig gut (Rothkotter et al., 199 1 ), but their actual role in the immune system is unknown. Within 1 week the number and distribution of cells in the lamina propria had altered dramatically. The increase in CD2+ cells after birth, and in particular the localisation of CD4+ cells in the villus lamina propria, as described by other workers (Rothkotter et al., 199 1; Bianchi et al., 1992) implies the rapid acquisition of the potential for induction of active immune responses within the lamina propria. This is further suggested by a parallel increase in numbers of class II bearing cells, also in the villus lamina propria. The expression of class II antigens may be constitutive or may be a consequence of local production of cytokines by activated T cells. However, the close association between the two cell types again suggests the capacity for presentation of environmental antigens within the lamina propria of the immature pig. The distribution of cells bearing the macrophage/granulocyte marker was different from that of the class II expressing cells, suggesting a role for these cells distinct from that proposed for class II+ antigenpresenting cells in villus. Their position in the crypts of unweaned animals is also apparently not consistent with a role as phagocytic cells. Following weaning, however, increases in macrophage/granulocyte cells were apparent within the villus lamina propria and these cells may be involved in local, non-specific defence. IL-2R+ cells were proportionately numerous in the lamina propria of newborn, presuckled piglets and some were found in the epithelium. Their morphology was consistent with that of blastoid cells with large cytoplasm and large nuclei, suggesting a high level of activity in the absence of major environmental challenge. Given the absence of antigenic stimulation in the newborn piglet, it is possible that these cells were undergoing antigen-independent activation. This is supported by the observation that the absolute number of IG2R+ cells remained constant in the lamina propria, but their proportion within the total cell population declined with age as the intestine became populated. Further, the numbers of IG2R+ cells was not increased in weaned piglets, despite the increase in CD2+ cells. Taken with the slight increase in class II expression in weaned animals this suggests that the increases in CD2+ T cells represented antigen-nonspecific recruitment of cells to the intestine as a result of cytokine release by a small number of antigen-reactive T cells, comparable to that seen in cutaneous DTH reactions ( Askenase and Van Loveren, 1983 ). As observed in newborn animals, the phenotype of these CD2+ cells appeared to be predominantly CD4-CD88. Since the changes following weaning were similar to those seen with age it is
326 h4.A. Vega-Lbpez et al. / VeterinaryImmunology and Immunopathology 44 (1995) 319-327
likely that, both in weaned and unweaned piglets, population of the intestine with immunological cells is driven by responses to gradual exposure to environmental (food and microbial) antigens. This would be consistent with the more limited population of the intestine seen in germ-free piglets (Rothkotter et al., 1991). However, the absence of an increase in IG2R+ cells, either with age or weaning, suggests that the majority of recruited cells are not active locally.
Acknowledgements
This work was supported by a linked research grant, LRG 18 from the Agricultural and Food Research Council. Dr Vega-Lopez was in receipt of a grant ( 54 19 1) from the Science and Technology National Council (CONACyT ), Mexico.
References Askenase, P.W. and van Loveren, H., 1983. Delayed-type hypersensitivity: activation of mast cells by antigen-specific T-cell factors initiates the cascade of cellular interactions. Immunol. Today, 4: 259-264. Bailey, M., Stevens, K., Bland, P.W. and Stokes, C.R., 1992. A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors. J. Immunol. Methods, 153: 85-9 1. Bianchi, A.T., Zwart, R.J., Jeurissen, S.H. and Moonen-Leusen, H.W., 1992. Development of the Band T-cell compartments in porcine lymphoid organs from birth to adult life: an immunohistological approach. Vet. Immunol. Immunopathol., 33: 201-22 1. Davis, W.C., Marusic, S., Lewin, H.A., Splitter, G.A., Perryman, L.E., McGuire, T.C. and Gorham, J.R., 1987. The identification and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens of the major histocompatibility complex for use in the study of the immune system of cattle and other species. Vet. Immunol. Immunopathol., 15: 337376. Gendelman, H.E., Moench, T.R., Narayan, 0. and Griffin, D.E., 1983. Selection of a fixative for identifying T cell subsets, B cells and macrophages in paraffin-embedded mouse spleen. J. Immunol. Methods, 65: 137-145. Hammerberg, C. and Schurig, G., 1986. Characterisation of monoclonal antibodies directed against swine leukocytes. Vet. Immunol. Immunopathol., 11: 107- 12 1. Hampson, D.J., 1986. Alterations in piglet small intestinal structure at weaning. Res. Vet. Sci., 40: 32-40. Hampson, D.J. and Kidder, D.E., 1986. Influence of creep feeding and weaning on brush border enzyme activities in the piglet small intestine. Res. Vet. Sci., 40: 24-3 1. Miller, B., Newby, T.J., Stokes, CR., Hampson, D. and Bourne, F.J., 1983. The role ofdietary antigen in the aetiology of post weaning diarrhoea. Ann. Rech. Vet., 14: 487-492. Miller, B.G., Newby, T.J., Stokes, C.R. and Boume, F.J., 1984. Influence of diet on postweaning malabsorption and diarrhoea in the pig. Res. Vet. Sci., 36: 187-193. Miller, B.G., James, P.S., Smith, M.W. and Boume, F.J., 1986. Effect of weaning on the capacity of pig intestinal villi to digest and absorb nutrients. J. Agric. Sci., 107: 579-589. Pescovitz, M.D., Lunney, J.K. and Sachs, D.H., 1985. Murine anti-swine T4 and T8 monoclonal antibodies: Distribution and effects on proliferative and cytotoxic T cells. J. Immunol., 134: 3744. Rothkotter, H.J., Ulbrich, H. and Pabst, R., 199 1. The postnatal development of gut lamina propria
M.A. Vega-Lbpez et al. / Veterinary Immunology and Irnrn~nupa~h~Io~ 44 (I 995) 319-327 327 lymphocytes: number, proliferation and T and B cell subsets in conventional and germ-free pigs. Pediatr. Res., 29: 231-242. Saalmuller, A., Hirt, W. and Reddehase, M.J., 1989. Phenotypic discrimination between thymic and extrathymic CD44CD8- and CD4+CD8+ porcine T lymphocytes. Eur. J. Immunol.. 19: 201 l2016. Vega-Lopez, M.A., Telemo, E., Bailey, M., Stevens, K. and Stokes, C.R., 1993. Immune cell distribution in the small intestine of the pig: immunohistological evidence for an organised compartmentalization in the lamina propria. Vet. Immunol. Immunopathol., 37: 49-60.
Veterinary Immunology and Immunopathology
and
immunopathology
44(1995)319-327
Effect of early weaning on the development of immune cells in the pig small intestine M.A. Vega-Lbpez”, M. Baileyb**, E. Telemob%*, CR. Stokesb ‘INIFAP-SARH, Mexico and FES-Cuautitlh-CM, Mexico bDivision of Molecular and Cellular Biology, Department of Clinical Veterinary Sciences, UniversityofBristol, Langford House, Langford, Bristol BS18 7011, UK
Accepted 9 February 1994
Abstract The controlled effects of age and weaning on the numbers of CD2 + T cells, subsets (CD4+, CD8+), accessory cells (macrophage/granulocyte) and cells expressing MHC class 11 (DQw ) and IL-2R in the piglet intestine was investigated. At birth low numbers of CD2+CD4_CD8cells were the only demonstrable T cells in the intestine. Monocyte/granulocyte and MHC class II+ cells were also detected in low numbers and IL-2R+ cells were proportionally quite numerous. All those cell populations, except the IL-2R+ cells, increased thereafter and peaked at Week 7 when the numbers of cells were comparable with those of adult animals. CD4+ cells increased dramatically after Week 1. In contrast, CD8+ remained scarce until after 5-7 weeks of age in unweaned animals. Four days after weaning at 3 weeks old, there were increases in CD2+ (PC 0.00 1) and macrophage/granulocyte (PC 0.0 1) cells in proximal small intestinal villi and in CD2+ cells only (PC 0.01) in crypts. No significant changes in cell numbers were demonstrated in the distal small intestine.
1. Introduction
At weaning, the young pig is abruptly subjected to a radical change in diet coupled with the waning of maternally derived serum immunoglobulins and the withdrawal of the locally protective elements of milk. The consequence of this * Corresponding author. ’ Present address: Clinical Immunology Department, University Hospital, Goteborg, 0165-2427/95/$09.50 0 1995 Elsevier Science B.V. AI1rights reserved SSDIOl65-2427(94)05309-G
Sweden.
320 M.A. Vega-L6pe.zet al. / Veterinary Immunology and Immunopathology 44 (1995) 319-327
may be to precipitate the onset of the syndrome of postweaning diarrhoea, characterised by villus atrophy, malabsorption due to enterocyte immaturity and bacterial overgrowth (Hampson, 1986; Hampson and Kidder, 1986). It has been postulated that an immune reaction (hypersensitivity) to fed protein may be involved in the early pathogenesis of this syndrome (Miller et al., 1983 ), predisposing to subsequent opportunistic infections (Escherichia co/i) which may prolong and exaggerate the situation (Miller et al., 1984). Because of the importance of enteric diseases to both animal and human health, the study of the immune system in the gut is crucial. We have recently reported a degree of organisation of the lymphoid cells within the diffuse lamina propria in mature pigs (VegaLopez et al., 1993). The appearance of such cells has been described by other workers in gnotobiotic piglets in low numbers after birth (Rothkotter et al., 199 1) and in piglets reared conventionally under systems involving weaning at 5 weeks of age (Bianchi et al., 1992). However, under the conditions of these experiments, the effects of weaning could not be separated from the effects of age and exposure to environment. In addition, postweaning disease is most severe in piglets weaned at 3 weeks of age. Here, we have examined changes in cells populating the intestine, in piglets maintained on the sow without access to solid food and in a controlled study after weaning. 2. Material and methods 2.1. Animals
All the. animals used were from a minimal disease herd of large white sows, maintained at the University of Bristol. Piglets suckling the sow were not allowed access to creep feed. Weaned piglets were fed a soya-based diet, the composition of which has been described (Miller et al., 1986). The animals were killed in accordance with local guidelines, with an overdose of sodium pentobarbitone (Euthatal, RMB Animal Health Ltd., Dagenham, UK). 2.2. Collection of samples
Samples from proximal ( 1 m from the pyloric junction) and distal ( 1 m from ileo-caecal junction) small intestine were either snap frozen in liquid nitrogen, or immediately fixed in paraformaldehyde-lysine-periodate fixative as described by Gendelman et al. ( 1983). Serial 2-4 pm sections of these tissues were prepared and used for immunohistochemical staining. 2.3. Monoclonal antibodies
Monoclonal antibodies recognising T cell markers and accessory cells were provided by Dr. J. Lunney, NIH Bethesda. The clones used were MSA4, anti-CD2 (Hammerberg and Schurig, 1986)) 74- 12-4, anti-CD4 and 76-2- 11, anti-CD8
M.A. Vega-L&e2 et al. / Veterinary Immunology and Immunopathology 44 (1995) 319-327
32 1
(Pescovitz et al., 1985) and 74-22-l 5, anti-monocyte/granulocyte (Hammerberg and Schurig, 1986). Clone H-42-A, recognising an MHC class II (DQw) determinant (Davis et al., 1987) was obtained from V.M.R.D., Washington. Clone 23 1.3B2, anti-IGZR, was generated in this laboratory (Bailey et al., 1992). 2.4. Immunohistochemistry The procedure used has been fully described by Vega-Mpez et al. ( 1993 ). Briefly, tissue sections were incubated with the optimal dilution of monoclonal antibody, then with an affinity purified, biotinylated rabbit anti-mouse antibody (Dakopatts, Glostrup, Denmark) followed by Streptavidin-horse radish peroxidase-avidin complex (Dakopatts). The reaction was visualised with 0.05% diaminobenzidine in 0.05 M Tris-HCl plus 0.01% hydrogen peroxide. The slides were counterstained with Harris haematoxylin and mounted in DPX. 2.5. Counts of cells Cells were counted using a high resolution, semi-automatic, computer assisted image analyser system (VIDS IV, Analytical Measuring Systems, Saffron Walden, UK). The positive cells were counted after the area in the lamina propria was calculated. The results were expressed as cells mm-*. At least five randomly selected fields in each tissue sample were counted, and because of the particular distribution of cells (Vega-Lopez et al,, 1993), the villus and crypt zones were counted separately. 2.4. Statistics The differences between groups were tested by analysis of variance using litter and treatment as blocks where appropriate. 3. Results 3. I. Changes in cell populations with age
Two litters of ten piglets each were selected and maintained on the sow without access to solid feed. Two piglets from each litter were killed at 0 (presuckled), 1, 3,5, and 7 weeks of age. The 6-month-old animals were described by Vega-Lopez et al. (1993). At birth, the few CD2+ cells were evenly distributed in the intestinal lamina propria. No staining for CD4/CD8 was apparent at this age and these cells were therefore CD4/CD8 negative (Fig. 1). Sites in the proximal and distal small intestine contained similar numbers of CD2+ cells. At all times after birth, these cells were more concentrated in villi than in crypts. The number of CD2+ cells in lamina propria and within the epithelium increased, reaching a maximum at
322 M.A. Vega-Lbpez et al. / Veterinary Immunology and Immunopathology 44 (1995) 319-327
Fig. 1. Intestine of newborn, presuckled piglet stained for CD2.
Weeks 5-7 (Fig. 2 (A) ). After the first week of age, an increase of CD4+ cells was also detectable within the lamina propria only. In contrast, CD8+ cells only moderately increased by the fifth week of age. The proportion of the CD2+ cells in the epithelial compartment increased from 38% at birth, to 53% at 7 weeks of age in the proximal small intestine, and up to 64% in the ileum. Low numbers of monocyte/granulocyte cells were present at birth with similar numbers of cells in the proximal small intestine and ileum (Fig. 2(B) ). These cells appeared to be evenly distributed in the lamina propria. After the first week of age, more cells were found in the distal than proximal small intestine. In addition the crypt zone was more populated than the villi. The number of cells reached a maximum at 5 weeks of age. IG2R+ cells were found in significant numbers at birth (Fig. 2 (C) ) but the numbers and distribution did not change significantly with age. These cells had large nuclei with prominent cytoplasm, and they were evenly distributed in the intestinal lamina propria. The numbers of MHC class II+ cells are shown in Fig. 2 (D). Even by 1 week of age, a dramatic increase in the number of positive cells was evident. The cells were concentrated in the villi, but significant numbers were also present in the crypts. Again, the density of cells peaked at Week 5 and this was most obvious in the villi. In the crypts, the number of positive cells remained quite stable with a transient drop in numbers at Week 3. Some of these cells showed dendritic morphology and were occasionally situated close to the epithelium. As previously reported, no staining for class II was detected in enterocytes.
en
2000
3000
4000
0
0
0
1
1
Weeks
3
3
7
7
6 months
6 months
\”
,”IA z 0
E 2
% E
2000
3000
4000
5000
0
0
1
WeeksofzkP
3
5
7
6 months
D
C
(A), the antigen recognised by the monoclonal antibody 74-22-l 5 (B), IL-2R (C) and MHC class II (D) piglets of increasing ages. Error bars are standard deviations of four animals.
of Age
5
5
Proljmal jetinal villi Pr‘,d,“al Jeju,d Crypts Distal jejunal wlli Distal jejunal crypts
Fig. 2. Mean numbers of cells expressingCD2 in intestinal lamina propria of litter-matched
c c-l
:: -z
E z
E
s
q
0
q q
2 z
324 M.A. Vega-Lbpez et al. I Veterinary Immunology and Immunopathology 44 (1995) 319-327
3.2. Eflect of weaning on cell populations
Two litters of ten piglets each were selected and at 2 1 days of age, live piglets from each litter were weaned onto a soya-containing diet. The remaining piglets were maintained on the sow without access to solid feed. All piglets were killed at 25 days of age. A comparison of weaned versus unweaned groups showed that in weaned animals there were significantly greater numbers of CD2+ (P
““““1T
E E iI? m 3
II
4000
T
1
CD2
MHC Class II
Cell surface
IL -2R
Mo/PMN
marker
Fig. 3. Mean numbers of cells expressing CD2, the antigen recognised by the monoclonal antibody 7422-15, MHC class II and IL-2R in villi (A) and cryps (B) of proximal jejunum of unweaned and 4 day weaned piglets. Error bars represent standard deviations of ten animals.
M.A. Vega-Lbpez et al. / Veterinary Immunology and Immunopathology
44 (1995) 319-327
325
4. Discussion The results observed in piglets of different ages are in agreement with those of other workers (Rothkotter et al., 199 1; Bianchi et al., 1992 ), but have been extended to include expression of the potentially modulatable antigens, MHC class II and interleukin 2 receptors. At birth, the majority of the T lymphocytes identified in the lamina propria of newborn animals lacked both the CD4 and CD8 markers. Many of these cells with unusual phenotype (CD2+CD4_CD8) were located in the epithelium. This dual-negative population of cells has been described in adult pig blood (Saalmuller et al., 1989), and recently in pig gut (Rothkotter et al., 199 1 ), but their actual role in the immune system is unknown. Within 1 week the number and distribution of cells in the lamina propria had altered dramatically. The increase in CD2+ cells after birth, and in particular the localisation of CD4+ cells in the villus lamina propria, as described by other workers (Rothkotter et al., 199 1; Bianchi et al., 1992) implies the rapid acquisition of the potential for induction of active immune responses within the lamina propria. This is further suggested by a parallel increase in numbers of class II bearing cells, also in the villus lamina propria. The expression of class II antigens may be constitutive or may be a consequence of local production of cytokines by activated T cells. However, the close association between the two cell types again suggests the capacity for presentation of environmental antigens within the lamina propria of the immature pig. The distribution of cells bearing the macrophage/granulocyte marker was different from that of the class II expressing cells, suggesting a role for these cells distinct from that proposed for class II+ antigenpresenting cells in villus. Their position in the crypts of unweaned animals is also apparently not consistent with a role as phagocytic cells. Following weaning, however, increases in macrophage/granulocyte cells were apparent within the villus lamina propria and these cells may be involved in local, non-specific defence. IL-2R+ cells were proportionately numerous in the lamina propria of newborn, presuckled piglets and some were found in the epithelium. Their morphology was consistent with that of blastoid cells with large cytoplasm and large nuclei, suggesting a high level of activity in the absence of major environmental challenge. Given the absence of antigenic stimulation in the newborn piglet, it is possible that these cells were undergoing antigen-independent activation. This is supported by the observation that the absolute number of IG2R+ cells remained constant in the lamina propria, but their proportion within the total cell population declined with age as the intestine became populated. Further, the numbers of IG2R+ cells was not increased in weaned piglets, despite the increase in CD2+ cells. Taken with the slight increase in class II expression in weaned animals this suggests that the increases in CD2+ T cells represented antigen-nonspecific recruitment of cells to the intestine as a result of cytokine release by a small number of antigen-reactive T cells, comparable to that seen in cutaneous DTH reactions ( Askenase and Van Loveren, 1983 ). As observed in newborn animals, the phenotype of these CD2+ cells appeared to be predominantly CD4-CD88. Since the changes following weaning were similar to those seen with age it is
326 h4.A. Vega-Lbpez et al. / VeterinaryImmunology and Immunopathology 44 (1995) 319-327
likely that, both in weaned and unweaned piglets, population of the intestine with immunological cells is driven by responses to gradual exposure to environmental (food and microbial) antigens. This would be consistent with the more limited population of the intestine seen in germ-free piglets (Rothkotter et al., 1991). However, the absence of an increase in IG2R+ cells, either with age or weaning, suggests that the majority of recruited cells are not active locally.
Acknowledgements
This work was supported by a linked research grant, LRG 18 from the Agricultural and Food Research Council. Dr Vega-Lopez was in receipt of a grant ( 54 19 1) from the Science and Technology National Council (CONACyT ), Mexico.
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