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Effect of colostral ingestion on immunoglobulin-positive cells in calves
Veterinary Immunology and Immunopathology 62 Ž1998. 51–64
Effect of colostral ingestion on immunoglobulin-positive cells in calves B.M. Aldridge, S.M. McGuirk, D.P. Lunn
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Department of Medical Sciences, School of Veterinary Medicine, UniÕersity of Wisconsin, 2015 Linden DriÕe West, Madison, WI 53706, USA Accepted 10 November 1997
Abstract The importance of colostrum for passive transfer of maternal immunoglobulin in calves is well established. Colostrum is thought to have additional generalized and antigen-specific immunomodulatory activities, of which the downregulation of endogenous immunoglobulin production is best documented. The objective of this study was to examine whether ingestion of colostrum altered the B cell subpopulations in the lymph nodes of newborn calves. Calves were fed one gallon of either fresh colostrum ŽGroup A, n s 5., milk replacer ŽGroup B, n s 5. or treated Žfrozen or irradiated. colostrum ŽGroup D, n s 4. and were euthanized at 36–48 h. An additional 5 calves ŽGroup C, 3 newborn and 2 mid-term fetuses. did not receive any feedings; the neonatal calves were euthanized immediately following birth. Mesenteric and regional lymph nodes from all calves were analyzed by immunocytochemistry using monoclonal antibodies recognizing bovine IgA, IgG1, IgG2, and IgM. Calves from Groups B and C Žcolostrum deprived, neonates, and fetuses. showed a consistent pattern of IgG1 and IgG2 positive cells scattered individually and in clusters throughout lymph node cortex, paracortex, and cortico–medullary junction. In sharp contrast, no IgG1 and IgG2 positive cells were present in the lymphoid tissues of colostrum fed calves ŽGroups A or D.. Numbers of IgM and IgA positive cells were similarly distributed in all calf groups. These findings demonstrate that colostrum feeding reduces the number of immunoglobulin positive cells in the lymphoid tissues of newborn calves in an isotype-specific manner. This results in the elimination of IgG1 and IgG2 positive cells that are present in both fetuses and newborn calves. This effect is not eliminated by freezing or irradiation, indicating that a
Abbreviations: IL: interleukin; TNF: tumor necrosis factor; Ig: immunoglobulin; sIg: surface immunoglobulin; MLN: mesenteric lymph node; OCT: optimal cutting temperature; HqE: hematoxylin–eosin; TBS: tris-buffered saline; AEC: 3-amino–9-ethylcarbazole; IVIG: intravenous immunoglobulin; SRID: single radial immunodiffusion ) Corresponding author. Tel.: q1 608 265 2671; fax: q1 608 265 8020; e-mail: [email protected] 0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 5 8 - X
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non-cellular, cold-stable colostral factor is responsible. Systemically distributed colostral proteins such as immunoglobulin or cytokines are the most likely mediators. The significance of this phenomenon in terms of colostral modulation of calf endogenous antibody production is discussed. q 1998 Elsevier Science B.V. Keywords: Calves; Immunity; Colostrum; B-cells; Immunohistochemistry; Immunomodulation
1. Introduction Colostrum is an important source of nutrition and passive immunity for the newborn calf ŽGay, 1965; Newby et al., 1982.. Immunoglobulins are a critical component of colostrum and provide the calf with passive protection against infectious disease until its own immune system is able to mount immune responses ŽBanks and McGuire, 1989.. Calves that do not ingest and absorb adequate levels of colostral immunoglobulin are highly susceptible to infectious disease during the first weeks of life ŽLogan et al., 1974; Wells et al., 1996.. In addition to its role as a source of passive immunity, colostrum also has potent immunomodulatory properties and can prevent calves from mounting active immune responses to specific antigens ŽMenanteau-Horta et al., 1985.. An important practical consequence of these immunosuppressive effects is that colostrum fed calves have impaired vaccine responses compared to colostrum-deprived calves ŽHusband and Lascelles, 1975.. Furthermore, colostrum can inhibit local and systemic antibody production in the newborn calf and colostrum-fed calves have lower peripheral blood lymphocyte blastogenic responses to T and B cell mitogens than colostrum-deprived calves ŽClover and Zarkower, 1980; Husband and Lascelles, 1975; Kimman et al., 1989.. These immunomodulatory properties of colostrum have also been demonstrated in vitro. Colostrum impairs the proliferation of human T cell lines by inhibiting the induction of interleukin 2 ŽIL2. ŽSambasivarao et al., 1996. and decreases antibody production in peripheral blood mononuclear cells ŽTorre and Oliver, 1988.. Colostrum contains a number of immunologically active elements that may effect calf immune responses, including immunoglobulin, cytokines ŽIL-2, TNF, IL-1, IL-6., non-immunoglobulin proteins Žlactoferrin., and leukocytes ŽSordillo et al., 1991; Ye and Yoshida, 1995; Riedel Caspari, 1993.. Which of these factors is responsible for the colostral effects on the calf immune system is unknown. The best documented aspect of colostral immunomodulation in the calf is the down-regulation of endogenous immunoglobulin production ŽBanks and McGuire, 1989; Logan et al., 1974.. This down-regulation appears to result in both antigen-specific and generalized inhibition of antibody production ŽBanks and McGuire, 1989.. Generalized inhibition is demonstrated by the fact that endogenous immunoglobulin production appears earlier, and reaches higher peak levels in colostrum-deprived calves ŽHusband and Lascelles, 1975; Logan et al., 1974.. Much of this inhibition has been attributed to colostral immunoglobulin, since suppression of antibody production is correlated with serum levels of colostral immunoglobulin ŽLogan, 1974.. Furthermore, if only one class of immunoglobulin is fed to a calf, then endogenous production of only that class will be impaired ŽLogan et al., 1974.. Antigen-specific inhibition is manifested in calves with
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high levels of circulating colostral antibody to a specific antigen, which do not mount antibody responses when exposed to this antigen ŽHeckert et al., 1991; Bradshaw and Edwards, 1996.. However, recent studies demonstrate that such antigen exposure, while not producing detectable rises in antibody concentration, does prime the immune system for an anamnestic response on subsequent antigen exposure ŽBrar et al., 1978; Menanteau-Horta et al., 1985.. The maturation of B lymphocytes to antibody-producing plasma cells occurs through a series of changes that can be differentiated by surface immunoglobulin ŽsIg. expression ŽJaneway and Travers, 1997a.. Early B cell development through the pro- and pre-B cell stages is associated with rearrangement of the immunoglobulin heavy and light chain genes, respectively. Although the m chain is transiently expressed on large pre-B cells in combination with surrogate light chain as a part of the pre-B cell receptor, the first complete surface immunoglobulin is IgM on the surface of immature B cells. B cell development to this point occurs in the bone marrow and, after selection for self-tolerance, B cells undergo further maturation and recirculate through secondary lymphoid tissues. When mature B cells subsequently recognize foreign antigen and receive appropriate T-helper cell costimulatory signals, they become activated, undergoing heavy chain isotype switching and somatic hypermutation of both light and heavy chain genes. This results in the production of both sIg of the IgG, IgA or IgE isotypes. Therefore, studies of the number, distribution, and isotype of sIg-positive B cells in lymphoid organs can yield considerable information about the maturity of B cell populations and development of antibody responses. In this study, we examine the effect of colostral ingestion on the B cell populations of lymph nodes in calves by comparing the number and isotype of immunoglobulin-positive cells in the mesenteric and regional lymph nodes of colostrum-fed and colostrum-deprived calves. The aims of this study are to determine the effect of colostrum on local and systemic B cell population dynamics, and to provide evidence for the nature of colostral immunomodulatory factors.
2. Materials and methods 2.1. CalÕes A total of 19 Holstein calves were separated from their mothers immediately after birth. Group A calves Ž n s 5. were bottle-fed 1 gal of dam’s fresh colostrum within 6 h of birth, while Group B calves Ž n s 5. were fed 1 gal of commercial milk replacer. Group C calves Ž n s 5. did not receive any feedings and included three full-term calves that were euthanized within 6 h of birth by intravenous pentobarbitone injection, and two mid-term fetuses collected from fresh slaughtered cows Ž4–6 months gestational age.. Group D calves Ž n s 4. were fed treated colostrum; one calf was fed fresh colostrum from its own dam, which had been frozen to y708C, thawed slowly and fed within 6 h of birth. Two other Group D calves received similarly frozen colostrum from unrelated dams. The fourth calf in Group D was fed colostrum from its own dam, which was first treated by g-irradiation Ž2000 rads. to fragment the DNA of colostral cells.
54 B.M. Aldridge et al.r Veterinary Immunology and Immunopathology 62 (1998) 51–64 Fig. 1. Immunoglobulin positive cells in the mesenteric lymph node cortices of 36-h old calves from group B, colostrum-deprived Ža, b, c, and d. or group A, colostrum-fed Že, f, g, and h. Žmagnification 100=.. Sections are immunoperoxidase stained using mAbs recognizing bovine IgG1 Ža and e., IgG2 Žb and f., IgA Žc and g. and IgM Žd and h.. No IgG1 or IgG2 positive cells are detectable in lymph nodes from colostrum-fed calves. Colostrum-deprived calves show a consistent pattern of individual and grouped IgG1 and IgG2 positive cells throughout the cortex, paracortex and cortico–medullary junction. The number and distribution of IgA and IgM cells is similar between colostrum-fed and colostrum-deprived calves.
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Fig. 1 Žcontinued..
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Following the initial feeding, calves in groups A, B, and D were fed 2 l of commercial milk replacer twice daily and were euthanized at 36 h of age. Blood samples were collected immediately before euthanasia from all calves to measure plasma IgG levels as an indicator of colostral ingestion and colostral immunoglobulin absorption. IgG levels were measured by single radial immunodiffusion ŽIgG SRID kit, VMRD, Pullman, WA.. 2.2. Tissue preparation A full postmortem was performed on each calf immediately following euthanasia and tissues were collected for immunohistological examination. Blocks of mesenteric lymph node ŽMLN. were collected from the proximal, mid-, and distal jejunal and ileal mesentery. Prefemoral and prescapular lymph nodes were also collected. Sampling sites were standardized between individuals by anatomical comparisons. Tissue blocks were frozen in optimal cutting temperature embedding compound ŽOCT, Tissue Teke, Baxter McGaw Park, IL. over liquid nitrogen and stored at y708C prior to cutting 6–8 m m cryostat sections onto ProbeOne ŽFisher, Pittsburgh, PA. glass slides. To minimize any variation due to regional differences within and between lymph nodes, hematoxylinreosin ŽH q E. stained sections of the nodes were examined and the tissue blocks were oriented to produce sections consisting of at least 50% cortex, 25% medulla, and showing an intact Ž) 75% section circumference. capsule. To validate our results, section-cutting and staining were repeated at least twice for each lymph node examined. 2.3. Tissue examination Tissue sections were air dried at room temperature for 24–48 h. Prior to staining, slides were fixed in acetone and rehydrated in tris-buffered saline ŽTBS.. Staining was performed using the Vectastain Elite ABC kit ŽVector Laboratories, Burlingame, CA.. With the exception of the overnight step, all incubations were carried out at room temperature and slides were washed in TBS between each incubation. Following rehydration, tissue sections were preincubated with blocking serum for 20 min. Monoclonal antibody supernatants were applied to sections, and incubated at 48C overnight in a moist box. The antibodies used recognized the bovine lymphocyte markers CD3 Žcell line MM1A, VMRD., IgA, IgG1, IgG2 and IgM Žfrom the respective hybridoma lines M23, M37,M33, and M67, which were provided by Dr. Klaus Nielson, Agriculture Canada ŽNielson and Henning, 1990.. Slides were subsequently incubated with biotinylated goat anti-murine IgG followed by the Vectastain Elite ABC avidin linked peroxidase reagent for 30 min each. Finally, slides were incubated for 5 min in AEC Ž3-amino–9-ethylcarbazole. substrate solution ŽAEC chromogen kit, Sigma, St. Louis, MO. and counterstained in hematoxylin prior to mounting using Aquamount ŽLerner Laboratories, Pittsburgh, PA.. Each staining procedure contained an isotype-matched negative-control antibody, and sections incubated with just secondary antibody were also examined. Sections from all groups were stained simultaneously to minimize inter-assay variation. Each section was
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examined by light microscopy. A semi-quantitative estimation of the number of positive staining cells was made for each tissue section. The sections were examined under medium Ž=100. power and the number of regions containing cells with surface-specific positive staining was categorically graded as absent Ž0., low Ž1., medium Ž2. or high Ž3.. Examinations were directed primarily towards the cortical lymphoid tissue, since this region contains the majority of lymph node immunoglobulin-positive B cell populations.
3. Results Group A colostrum-fed calves had mean plasma IgG levels of 1840 mgrdl Žrange 520–3100 mgrdl. while IgG was undetectable in group B colostrum-deprived calves analyzed by the same technique. Group D calves receiving treated colostrum also achieved successful passive immunoglobulin transfer Žmean IgG 1090 mgrdl, range 680–1500 mgrdl.. All calves were healthy by gross pathological examination. Lymph node sections stained with H q E showed a similar architecture among all calf groups. There were some structural differences between the nodes of these calves and those reported for adult cattle ŽBanks, 1993., in that the cortex was fairly homogenous, with indistinct primary and secondary lymphoid follicles. In none of the calf lymph node sections examined was there any evidence of primary germinal centers. For CD3 and all Ig isotypes except IgA, the number and distribution of positive-staining cells was the same in the mesenteric and peripheral lymph nodes of the same calf. IgA positive cells were commonly found in mesenteric lymph nodes, but were very rare in peripheral lymph nodes. Results are therefore given for mesenteric lymph nodes. All lymph nodes of all calf groups showed extensive regions of cortical CD3 positive cells corresponding to T cell regions. The most abundant immunoglobulin-bearing cell in the lymph nodes of all calf groups were IgM-positive cells ŽFig. 1.. There were no detectable differences in number or distribution of IgM-positive cells among newborn calves, although numbers were slightly reduced in the Group C fetuses. The IgM-positive cells were present predominantly in well-defined islands in the lymphoid cortex, but could also be seen scattered individually in the paracortex and cortico–medullary junction. The density of positive-staining cells varied between calves, but there was no
Table 1 Comparison of immunoglobulin-bearing cells in mesenteric lymphoid tissues from group A–D calves
Group A Žfresh colostrum. Group B Žcolostrum-deprived. Group C Neonates Fetuses Group D Žtreated colostrum.
IgG1
IgG2
IgA
IgM
0 2
0 2
0–1 1
3 3
1–2 1 0
1–2 1 0
0–1 1 0–1
3 2 3
The number of cells with positive staining was categorically graded as absent Ž0., low Ž1., medium Ž2. or high Ž3.. A range of density is given where cell numbers varied among individuals in a group.
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recognizable pattern of difference between calf groups. The number of IgM-positive regions per medium powered field and the density of IgM-positive cells per region was always grade 2 to 3 ŽTable 1.. The regions containing IgM-positive cells were equally abundant in mesenteric and peripheral lymph nodes. The density and abundance of IgA-positive cells was also consistent between calves ŽTable 1.. These IgA-positive cells were much less abundant than IgM-positive cells and usually were present as individual cells scattered throughout the cortex, paracortex, and cortico–medullary junction ŽFig. 1.. Occasional aggregates of IgA-positive cells were observed in all groups. IgA-positive cells were very rare in peripheral lymph nodes in any calf group. The major differences between calf groups were confined to the IgG1 and IgG2 positive cells ŽTable 1 and Fig. 1.. In Group A, calves fed fresh colostrum, no IgG1 or IgG2 positive cells were observed in any sections from either mesenteric or peripheral lymph nodes. In contrast, the lymph nodes of colostrum-deprived Group B calves contained numerous IgG1 and IgG2 positive cells, with a similar pattern of distribution and density. The IgG positive cells were frequently present in clusters in cortical regions compatible with the location of lymphoid follicles. Discrete, individual, and small clusters of IgG positive cells were also scattered throughout the cortex, paracortex, and at the cortico–medullary junction. Group C calves were examined to determine what Ig positive cell populations were present prior to colostral ingestion. In the two neonatal Group C calves, IgA and IgM positive cells were present in similar numbers and with a similar distribution to other post-natal calves ŽGroups A, B, and D.. In Group C fetuses, IgA and IgM positive cells were present but in reduced numbers when compared to group A calves. Lymphoid tissues from Group C neonatal calves contained IgG1 and IgG2 positive cells in similar numbers to Group B calves ŽFig. 2.. Similarly, fetuses contained IgG positive cells, although their numbers were markedly reduced. In Group D calves, which received frozen or irradiated colostrum, the numbers and distribution of Ig positive cells in lymphoid tissues were indistinguishable from the Group A calves that received fresh colostrum. 4. Discussion Colostrum is principally regarded as a source of passive immunity in the newborn calf. This study demonstrated that colostrum also has a profound effect on postnatal development of the calf’s endogenous immune system and specifically on the B cell lineage. Examination of mid-term fetuses, neonatal calves, and 36-h old colostrum-deprived calves demonstrated the increasing presence of sIg-positive B cells of all isotypes at each stage. However, ingestion of colostrum changed the number of sIg-positive cells Fig. 2. Immunoglobulin positive cells in the mesenteric lymph nodes of group C newborn calves, 6 h after birth Žmagnification 100=.. Sections are immunoperoxidase stained using mAbs recognizing surface IgG1 Ža., IgG2 Žb., IgA Žc. and IgM Žd.. Numerous IgG1 and IgG2 positive cells are seen throughout the cortex, paracortex and cortico–medullary junction indicating the presence of these cells prior to the ingestion of colostrum. The number and distribution of IgA and IgM cells is similar to those seen in Fig. 1.
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in lymph nodes in an isotype-specific manner resulting in the disappearance of IgG1 and IgG2-positive lymph node cells alone. This observation is consistent with elimination of the IgG-positive B cells that were present at birth, or the downregulation of sIgG expression by these cells. The presence of sIg-positive cells of various isotypes in both fetuses and term calves is consistent with the previously reported ontogeny of antibody production in calves ŽOsburn et al., 1982.. Similarly, the distribution of IgM-positive cells was consistent with the distribution of B-cell areas described in calves and older cattle ŽGaleotti et al., 1993.. The absence of distinct primary and secondary lymphoid follicles and abundance of IgM-positive cells compared to other isotypes may reflect the antigen naivete´ of calves in this study. The correlation of sIg expression with developmental stage has been defined in mice and humans ŽJaneway and Travers, 1997a.. The scattered cortical distribution of IgG and IgA-positive cells seen in fetuses and colostrum-deprived calves in this study is consistent with the description of mature B cells that have recently encountered antigen, interacted with T cells, and undergone antigen-driven class-switching prior to migrating to follicles and establishing germinal centers ŽJaneway and Travers, 1997b.. The presence of IgG-positive and IgA-positive cells in fetuses and colostrum-deprived calves indicates that isotype switching occurs in some B cells before birth. This switch may occur as a result of an in utero exposure to maternal commensal microbes ŽSchultz et al., 1973., or spontaneously, as described for natural or pre-immune antibody production in other neonates ŽCukrowska et al., 1995.. These natural antibodies are produced independent of exposure to environmental antigens, are polyspecific, have low affinity, react with a variety of self-antigens, and are spontaneously formed ŽCukrowska et al., 1996.. The function of natural antibodies is not clear, but they may represent a first line of defense during fetal and neonatal life. In Group D calves, neither a rapid freeze–thaw cycle or g-irradiation prevented colostral ingestion from eliminating sIgG-positive cells in lymph nodes. This finding tends to exclude colostral cells and cold-labile factors as causes of this phenomenon, despite the fact that both of these factors have been shown to affect immunoglobulin production in newborn calves and pigs, respectively ŽRiedel Caspari and Schmidt, 1991; Riedel Caspari, 1993; Klobasa et al., 1990.. Furthermore, changes in IgG-positive cells were not confined to intestinally-associated lymphoid tissues, but were observed in all lymph nodes examined. This indicates that the colostral element was an absorbable, widely distributed, cold-stable factor such as immunoglobulin or a cytokine. Immunoglobulins may be the most likely candidate given their established effect on endogenous antibody production. Colostrum-associated suppression of antibody production correlates with the level of circulating antibody and is isotype-specific ŽHeckert et al., 1991; Husband and Lascelles, 1975; Van Zaane et al., 1986.. In addition, immunoglobulins are cold-resistant, systemically absorbed, and distributed to all tissues ŽBoyd and Boyd, 1987; Haines et al., 1992; Besser, 1993.. A number of potential mechanisms could explain why IgG-positive cells were absent from the lymph nodes of colostrum-fed calves. These include downregulation due to capping and endocytosis of sIg, altered trafficking or the death and removal of sIg-positive cells. Downregulation of sIg expression can occur in the presence of anti-immunoglobulin antibody, and is reversible immediately when the anti-immuno-
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globulin is removed ŽLawton and Cooper, 1979.. This mechanism could explain the findings in this study since colostrum can contain anti-idiotypic antibodies and B cells of the newborn are susceptible to sIg modulation ŽCollins et al., 1991; Raff et al., 1975.. In mice, anti-immunoglobulin antibodies reduce sIg expression in B cells by inducing capping and endocytosis ŽLawton and Cooper, 1979.. In neonatal B cells, this process is irreversible and results in functional inactivation ŽKearney et al., 1978; Raff et al., 1975.. Since colostrum can contain antibodies with extensive anti-immunoglobulin specificities, this mechanism may explain the changes in sIg expression seen in this study ŽCollins et al., 1991.. Passive transfer of antibodies has also been shown to affect B cell populations in the context of intravenous immunoglobulin ŽIVIG. therapy, and can alter an individual’s antibody repertoire by eliminating certain bone marrow B cell lineages or modifying emergent B cell repertoires ŽSunblad, 1994.. It is therefore possible that colostral immunoglobulin changed sIgG-expression by interacting with B cells directly, or by altering T cell function ŽSunblad, 1994.. The cellular mechanisms by which immunoglobulin-induced changes in antibody production are induced are often mediated by cytokines ŽAbe et al., 1994; Andersson et al., 1994; Toyoda et al., 1994.. Alternatively, it remains possible that bovine colostral cytokines ŽSordillo et al., 1991., or other non-immunoglobulin proteins ŽSambasivarao et al., 1996., may have directly down-regulated sIg expression in this study. A major immunological transition occurs at the time of birth, as the neonate is exposed to a large number of foreign antigens in its new environment. The colostrum-induced immunomodulation described in this study may provide a biological advantage during this process. Because colostrum ingestion eliminated only IgG-positive B cells, less mature sIgM-positive cells, and IgA-positive cells in mesenteric lymph nodes with a probable role in mucosal immune responses were spared. This confined the effect to a B cell population most likely to produce IgG antibodies restricted to the circulating and systemic compartments. This may be consistent with suppression of endogenous antibody responses in the calf to circulating passively transferred maternal components, such as immunoglobulins themselves. Overall, this may result in tolerance of maternal immunoglobulins, and an increased longevity of these passively transferred antibodies. Alternatively, suppression of immunoglobulin production in circulating and systemic compartments may also allow the passage of colostral immune cells to the calf. There is indirect evidence that colostral cells upregulate antigen-specific immune responses in the calf, and definitive evidence for the transfer of immune cells between dam and offspring in other species ŽArchambault et al., 1988; Ellis et al., 1996; Le Jan, 1996; Riedel Caspari, 1993; Sheldrake and Husband, 1985; Williams, 1993.. Another potential immunomodulatory role for colostral antibodies may be influencing the calf antibody repertoire through idiotyperanti-idiotype interactions ŽKeller et al., 1991; Wikler, 1980.. This effect has been demonstrated in mice where neonates receiving antibodies bearing certain idiotypes were protected against infection, and were also primed for higher antigen-specific antibody responses on subsequent immunizations ŽOkamoto, 1991.. It is possible that the elimination of IgG-positive cells may result from such idiotyperanti-idiotype interactions. While any advantage of the elimination of IgG-positive B cells in calf lymph nodes subsequent to colostral ingestion remains uncertain, this is a further example of the
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immunomodulatory effects of colostrum in calves. An increased understanding of such interactions will have important implications for our understanding of the full immunological role of colostrum, and for our capacity to influence calf immune responses by active or passive vaccination.
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Husband, A.J., Lascelles, A.K., 1975. Antibody responses to neonatal immunisation in calves. Res. Vet. Sci. 18, 201–207. Janeway, C.A., Travers, P., 1997a. The development of B lymphocytes. In: Janeway, C.A., Travers, P., Hunt, S., Walport, M. ŽEds.., Immunobiology: The Immune System in Health and Disease, 3rd edn. Current Biology, London, pp. 5–3. Janeway, C.A., Travers, P., 1997b. The humoral immune response. In: Janeway, C.A., Travers, P., Hunt, S., Walport, M. ŽEds.., Immunobiology: The Immune System in Health and Disease, 3rd edn. Current Biology, London, pp. 5–8. Kearney, J.F., Klein, J., Bockman, D.E., 1978. B cell differentiation induced by lipopolysaccharide: V. Suppression of plasma cell maturation by anti-m: mode of action and characteristics of suppressed cells. J. Immunol. 120, 158. Keller, M.A., Palmer, C.J., Jelonek, M.T., Song, C.H., Miller, A., Sercarz, E.E., Calandra, G.B., Brust, J.L., 1991. Modulation of the immune response by maternal antibody. In: Mestecky, J. et al. ŽEds.., Immunology of Milk and the Neonate. Plenum, New York, pp. 207–213. Kimman, T.G., Westenbrink, F., Straver, P.J., 1989. Priming for local and systemic antibody memory responses to bovine respiratory syncytial virus: effect of amount of virus, virus replication, route of administration and maternal antibodies. Vet. Immunol. Immunopathol. 22, 145–160. Klobasa, F., Butler, J.E., Habe, F., 1990. Maternal–neonatal immunoregulation: suppression of de novo synthesis of IgG and IgA, but not IgM, in neonatal pigs by bovine colostrum, is lost upon storage. Am. J. Vet. Res. 51, 1407–1412. Lawton, A.R., Cooper, M.D., 1979. B cell ontogeny: immunoglobulin genes and their expression. Pediatrics 64, 750–757. Le Jan, C., 1996. Cellular components of mammary secretions and neonatal immunity: a review. Vet. Res. 27, 403–417. Logan, E.F., 1974. Quantitative studies on serum immunoglobulin levels in suckled calves from birth to five weeks. Vet. Rec. 94, 367–370. Logan, E.F., Stenhouse, A., Ormrod, D.J., Penhale, W.J., 1974. The role of colostral immunoglobulins in intestinal immunity to enteric colibacillosis in the calf. Res. Vet. Sci. 17, 280–301. Menanteau-Horta, A.M., Ames, T.R., Johnson, D.W., Meiske, J.C., 1985. Effect of maternal antibody upon vaccination with infectious bovine rhinotracheitis and bovine virus diarrhea vaccines. Can. J. Comp. Med. 49, 10–14. Newby, T.J., Stokes, C.R., Bourne, F.J., 1982. Immunologic activities of milk. Vet. Immunol. Immunopathol. 3, 67–94. Nielson, K., Henning, D., 1990. Monoclonal antibodies in veterinary medicine. Gen. Eng. Biotechnol. 10, 14–18. Okamoto, Y., 1991. Maternal determinants of neonatal immune response: effect of anti-idiotype in the neonate. Adv. Exp. Med. Biol. 310, 215–222. Osburn, B., MacLachlan, N., Terrell, T., 1982. Ontogeny of the immune system. J.A.V.M.A. 181 Ž10., 1049–1052. Raff, M.C., Owen, J.J.T., Cooper, M.D., 1975. Differences in susceptibility of mature and immature mouse lymphocytes to anti-immunoglobulin induced immunoglobulin suppression in vitro: possible implications for B cell tolerance to self. J. Exp. Med. 142, 1052. Riedel Caspari, G., 1993. The influence of colostral lymphocytes on the course of an experimental Escherichia coli infection and serum antibodies in the calf. Vet. Immunol. Immunopathol. 35, 275–288. Riedel Caspari, G., Schmidt, F.W., 1991. The influence of colostral leukocytes on the immune system of the neonatal calf: II. Effects on passive and active immunization. Dtsch. Tierarztl. Wochenschr. 98, 190–194. Sambasivarao, D., Hooton, J., Dost, A., Paetkau, V., 1996. A novel immunosuppressive factor in bovine colostrum blocks activation of the interleukin 2 gene enhancer at the NFAT site. Biochem. Cell Biol. 74, 585–593. Schultz, R.D., Dunne, H.W., Heist, C.E., 1973. Ontogeny of the bovine immune response. Infect. Immun. 7, 981–991. Sheldrake, R.F., Husband, A.J., 1985. Intestinal uptake of intact maternal lymphocytes by neonatal rats and lambs. Res. Vet. Sci. 39, 10–15.
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Sordillo, L.M., Redmond, M.J., Campos, M., Warren, L., Babiuk, L.A., 1991. Cytokine activity in bovine mammary gland secretions during the periparturient period. Can. J. Vet. Res. 55, 298–301. Sunblad, A., 1994. Observations on the mode of action of normal immunoglobulin at high doses. Immunol. Rev. 139, 125–157. Torre, P.M., Oliver, S.P., 1988. Changes in blastogenic activity of bovine blood mononuclear cells throughout the nonlactating period. J. Dairy Sci. 71, 1078–1084. Toyoda, M., Zhang, X., Petrosian, A., Galera, O., Wang, S., Jordan, S., 1994. Modulation of immunoglobulin production and cytokine mRNA expression in peripheral blood mononuclear cells by intravenous immunoglobulin. J. Clin. Immunol. 14, 178–189. Van Zaane, D., Ijzerman, J., De Leeuw, P.W., 1986. Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody. Vet. Immunol. Immunopathol. 11, 45–63. Wells, S.J., Dargatz, D.A., Ott, S.L., 1996. Factors associated with mortality to 21 days of life in dairy heifers in the United States. Prev. Vet. Med. 53, 49–60. Wikler, M., 1980. Immunoregulatory role of maternal idiotypes. J. Exp. Med. 152, 10224–10234. Williams, P.P., 1993. Immunomodulating effects of intestinal absorbed maternal colostral leukocytes by neonatal pigs. Can. J. Vet. Res. 57, 1–8. Ye, X., Yoshida, S., 1995. Lactoperoxidase and lactoferrin: changes in post partum milk during bovine lactational disorders. Milchwissenschaft 50, 67–71.
Effect of colostral ingestion on immunoglobulin-positive cells in calves B.M. Aldridge, S.M. McGuirk, D.P. Lunn
)
Department of Medical Sciences, School of Veterinary Medicine, UniÕersity of Wisconsin, 2015 Linden DriÕe West, Madison, WI 53706, USA Accepted 10 November 1997
Abstract The importance of colostrum for passive transfer of maternal immunoglobulin in calves is well established. Colostrum is thought to have additional generalized and antigen-specific immunomodulatory activities, of which the downregulation of endogenous immunoglobulin production is best documented. The objective of this study was to examine whether ingestion of colostrum altered the B cell subpopulations in the lymph nodes of newborn calves. Calves were fed one gallon of either fresh colostrum ŽGroup A, n s 5., milk replacer ŽGroup B, n s 5. or treated Žfrozen or irradiated. colostrum ŽGroup D, n s 4. and were euthanized at 36–48 h. An additional 5 calves ŽGroup C, 3 newborn and 2 mid-term fetuses. did not receive any feedings; the neonatal calves were euthanized immediately following birth. Mesenteric and regional lymph nodes from all calves were analyzed by immunocytochemistry using monoclonal antibodies recognizing bovine IgA, IgG1, IgG2, and IgM. Calves from Groups B and C Žcolostrum deprived, neonates, and fetuses. showed a consistent pattern of IgG1 and IgG2 positive cells scattered individually and in clusters throughout lymph node cortex, paracortex, and cortico–medullary junction. In sharp contrast, no IgG1 and IgG2 positive cells were present in the lymphoid tissues of colostrum fed calves ŽGroups A or D.. Numbers of IgM and IgA positive cells were similarly distributed in all calf groups. These findings demonstrate that colostrum feeding reduces the number of immunoglobulin positive cells in the lymphoid tissues of newborn calves in an isotype-specific manner. This results in the elimination of IgG1 and IgG2 positive cells that are present in both fetuses and newborn calves. This effect is not eliminated by freezing or irradiation, indicating that a
Abbreviations: IL: interleukin; TNF: tumor necrosis factor; Ig: immunoglobulin; sIg: surface immunoglobulin; MLN: mesenteric lymph node; OCT: optimal cutting temperature; HqE: hematoxylin–eosin; TBS: tris-buffered saline; AEC: 3-amino–9-ethylcarbazole; IVIG: intravenous immunoglobulin; SRID: single radial immunodiffusion ) Corresponding author. Tel.: q1 608 265 2671; fax: q1 608 265 8020; e-mail: [email protected] 0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 5 8 - X
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non-cellular, cold-stable colostral factor is responsible. Systemically distributed colostral proteins such as immunoglobulin or cytokines are the most likely mediators. The significance of this phenomenon in terms of colostral modulation of calf endogenous antibody production is discussed. q 1998 Elsevier Science B.V. Keywords: Calves; Immunity; Colostrum; B-cells; Immunohistochemistry; Immunomodulation
1. Introduction Colostrum is an important source of nutrition and passive immunity for the newborn calf ŽGay, 1965; Newby et al., 1982.. Immunoglobulins are a critical component of colostrum and provide the calf with passive protection against infectious disease until its own immune system is able to mount immune responses ŽBanks and McGuire, 1989.. Calves that do not ingest and absorb adequate levels of colostral immunoglobulin are highly susceptible to infectious disease during the first weeks of life ŽLogan et al., 1974; Wells et al., 1996.. In addition to its role as a source of passive immunity, colostrum also has potent immunomodulatory properties and can prevent calves from mounting active immune responses to specific antigens ŽMenanteau-Horta et al., 1985.. An important practical consequence of these immunosuppressive effects is that colostrum fed calves have impaired vaccine responses compared to colostrum-deprived calves ŽHusband and Lascelles, 1975.. Furthermore, colostrum can inhibit local and systemic antibody production in the newborn calf and colostrum-fed calves have lower peripheral blood lymphocyte blastogenic responses to T and B cell mitogens than colostrum-deprived calves ŽClover and Zarkower, 1980; Husband and Lascelles, 1975; Kimman et al., 1989.. These immunomodulatory properties of colostrum have also been demonstrated in vitro. Colostrum impairs the proliferation of human T cell lines by inhibiting the induction of interleukin 2 ŽIL2. ŽSambasivarao et al., 1996. and decreases antibody production in peripheral blood mononuclear cells ŽTorre and Oliver, 1988.. Colostrum contains a number of immunologically active elements that may effect calf immune responses, including immunoglobulin, cytokines ŽIL-2, TNF, IL-1, IL-6., non-immunoglobulin proteins Žlactoferrin., and leukocytes ŽSordillo et al., 1991; Ye and Yoshida, 1995; Riedel Caspari, 1993.. Which of these factors is responsible for the colostral effects on the calf immune system is unknown. The best documented aspect of colostral immunomodulation in the calf is the down-regulation of endogenous immunoglobulin production ŽBanks and McGuire, 1989; Logan et al., 1974.. This down-regulation appears to result in both antigen-specific and generalized inhibition of antibody production ŽBanks and McGuire, 1989.. Generalized inhibition is demonstrated by the fact that endogenous immunoglobulin production appears earlier, and reaches higher peak levels in colostrum-deprived calves ŽHusband and Lascelles, 1975; Logan et al., 1974.. Much of this inhibition has been attributed to colostral immunoglobulin, since suppression of antibody production is correlated with serum levels of colostral immunoglobulin ŽLogan, 1974.. Furthermore, if only one class of immunoglobulin is fed to a calf, then endogenous production of only that class will be impaired ŽLogan et al., 1974.. Antigen-specific inhibition is manifested in calves with
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high levels of circulating colostral antibody to a specific antigen, which do not mount antibody responses when exposed to this antigen ŽHeckert et al., 1991; Bradshaw and Edwards, 1996.. However, recent studies demonstrate that such antigen exposure, while not producing detectable rises in antibody concentration, does prime the immune system for an anamnestic response on subsequent antigen exposure ŽBrar et al., 1978; Menanteau-Horta et al., 1985.. The maturation of B lymphocytes to antibody-producing plasma cells occurs through a series of changes that can be differentiated by surface immunoglobulin ŽsIg. expression ŽJaneway and Travers, 1997a.. Early B cell development through the pro- and pre-B cell stages is associated with rearrangement of the immunoglobulin heavy and light chain genes, respectively. Although the m chain is transiently expressed on large pre-B cells in combination with surrogate light chain as a part of the pre-B cell receptor, the first complete surface immunoglobulin is IgM on the surface of immature B cells. B cell development to this point occurs in the bone marrow and, after selection for self-tolerance, B cells undergo further maturation and recirculate through secondary lymphoid tissues. When mature B cells subsequently recognize foreign antigen and receive appropriate T-helper cell costimulatory signals, they become activated, undergoing heavy chain isotype switching and somatic hypermutation of both light and heavy chain genes. This results in the production of both sIg of the IgG, IgA or IgE isotypes. Therefore, studies of the number, distribution, and isotype of sIg-positive B cells in lymphoid organs can yield considerable information about the maturity of B cell populations and development of antibody responses. In this study, we examine the effect of colostral ingestion on the B cell populations of lymph nodes in calves by comparing the number and isotype of immunoglobulin-positive cells in the mesenteric and regional lymph nodes of colostrum-fed and colostrum-deprived calves. The aims of this study are to determine the effect of colostrum on local and systemic B cell population dynamics, and to provide evidence for the nature of colostral immunomodulatory factors.
2. Materials and methods 2.1. CalÕes A total of 19 Holstein calves were separated from their mothers immediately after birth. Group A calves Ž n s 5. were bottle-fed 1 gal of dam’s fresh colostrum within 6 h of birth, while Group B calves Ž n s 5. were fed 1 gal of commercial milk replacer. Group C calves Ž n s 5. did not receive any feedings and included three full-term calves that were euthanized within 6 h of birth by intravenous pentobarbitone injection, and two mid-term fetuses collected from fresh slaughtered cows Ž4–6 months gestational age.. Group D calves Ž n s 4. were fed treated colostrum; one calf was fed fresh colostrum from its own dam, which had been frozen to y708C, thawed slowly and fed within 6 h of birth. Two other Group D calves received similarly frozen colostrum from unrelated dams. The fourth calf in Group D was fed colostrum from its own dam, which was first treated by g-irradiation Ž2000 rads. to fragment the DNA of colostral cells.
54 B.M. Aldridge et al.r Veterinary Immunology and Immunopathology 62 (1998) 51–64 Fig. 1. Immunoglobulin positive cells in the mesenteric lymph node cortices of 36-h old calves from group B, colostrum-deprived Ža, b, c, and d. or group A, colostrum-fed Že, f, g, and h. Žmagnification 100=.. Sections are immunoperoxidase stained using mAbs recognizing bovine IgG1 Ža and e., IgG2 Žb and f., IgA Žc and g. and IgM Žd and h.. No IgG1 or IgG2 positive cells are detectable in lymph nodes from colostrum-fed calves. Colostrum-deprived calves show a consistent pattern of individual and grouped IgG1 and IgG2 positive cells throughout the cortex, paracortex and cortico–medullary junction. The number and distribution of IgA and IgM cells is similar between colostrum-fed and colostrum-deprived calves.
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Fig. 1 Žcontinued..
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Following the initial feeding, calves in groups A, B, and D were fed 2 l of commercial milk replacer twice daily and were euthanized at 36 h of age. Blood samples were collected immediately before euthanasia from all calves to measure plasma IgG levels as an indicator of colostral ingestion and colostral immunoglobulin absorption. IgG levels were measured by single radial immunodiffusion ŽIgG SRID kit, VMRD, Pullman, WA.. 2.2. Tissue preparation A full postmortem was performed on each calf immediately following euthanasia and tissues were collected for immunohistological examination. Blocks of mesenteric lymph node ŽMLN. were collected from the proximal, mid-, and distal jejunal and ileal mesentery. Prefemoral and prescapular lymph nodes were also collected. Sampling sites were standardized between individuals by anatomical comparisons. Tissue blocks were frozen in optimal cutting temperature embedding compound ŽOCT, Tissue Teke, Baxter McGaw Park, IL. over liquid nitrogen and stored at y708C prior to cutting 6–8 m m cryostat sections onto ProbeOne ŽFisher, Pittsburgh, PA. glass slides. To minimize any variation due to regional differences within and between lymph nodes, hematoxylinreosin ŽH q E. stained sections of the nodes were examined and the tissue blocks were oriented to produce sections consisting of at least 50% cortex, 25% medulla, and showing an intact Ž) 75% section circumference. capsule. To validate our results, section-cutting and staining were repeated at least twice for each lymph node examined. 2.3. Tissue examination Tissue sections were air dried at room temperature for 24–48 h. Prior to staining, slides were fixed in acetone and rehydrated in tris-buffered saline ŽTBS.. Staining was performed using the Vectastain Elite ABC kit ŽVector Laboratories, Burlingame, CA.. With the exception of the overnight step, all incubations were carried out at room temperature and slides were washed in TBS between each incubation. Following rehydration, tissue sections were preincubated with blocking serum for 20 min. Monoclonal antibody supernatants were applied to sections, and incubated at 48C overnight in a moist box. The antibodies used recognized the bovine lymphocyte markers CD3 Žcell line MM1A, VMRD., IgA, IgG1, IgG2 and IgM Žfrom the respective hybridoma lines M23, M37,M33, and M67, which were provided by Dr. Klaus Nielson, Agriculture Canada ŽNielson and Henning, 1990.. Slides were subsequently incubated with biotinylated goat anti-murine IgG followed by the Vectastain Elite ABC avidin linked peroxidase reagent for 30 min each. Finally, slides were incubated for 5 min in AEC Ž3-amino–9-ethylcarbazole. substrate solution ŽAEC chromogen kit, Sigma, St. Louis, MO. and counterstained in hematoxylin prior to mounting using Aquamount ŽLerner Laboratories, Pittsburgh, PA.. Each staining procedure contained an isotype-matched negative-control antibody, and sections incubated with just secondary antibody were also examined. Sections from all groups were stained simultaneously to minimize inter-assay variation. Each section was
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examined by light microscopy. A semi-quantitative estimation of the number of positive staining cells was made for each tissue section. The sections were examined under medium Ž=100. power and the number of regions containing cells with surface-specific positive staining was categorically graded as absent Ž0., low Ž1., medium Ž2. or high Ž3.. Examinations were directed primarily towards the cortical lymphoid tissue, since this region contains the majority of lymph node immunoglobulin-positive B cell populations.
3. Results Group A colostrum-fed calves had mean plasma IgG levels of 1840 mgrdl Žrange 520–3100 mgrdl. while IgG was undetectable in group B colostrum-deprived calves analyzed by the same technique. Group D calves receiving treated colostrum also achieved successful passive immunoglobulin transfer Žmean IgG 1090 mgrdl, range 680–1500 mgrdl.. All calves were healthy by gross pathological examination. Lymph node sections stained with H q E showed a similar architecture among all calf groups. There were some structural differences between the nodes of these calves and those reported for adult cattle ŽBanks, 1993., in that the cortex was fairly homogenous, with indistinct primary and secondary lymphoid follicles. In none of the calf lymph node sections examined was there any evidence of primary germinal centers. For CD3 and all Ig isotypes except IgA, the number and distribution of positive-staining cells was the same in the mesenteric and peripheral lymph nodes of the same calf. IgA positive cells were commonly found in mesenteric lymph nodes, but were very rare in peripheral lymph nodes. Results are therefore given for mesenteric lymph nodes. All lymph nodes of all calf groups showed extensive regions of cortical CD3 positive cells corresponding to T cell regions. The most abundant immunoglobulin-bearing cell in the lymph nodes of all calf groups were IgM-positive cells ŽFig. 1.. There were no detectable differences in number or distribution of IgM-positive cells among newborn calves, although numbers were slightly reduced in the Group C fetuses. The IgM-positive cells were present predominantly in well-defined islands in the lymphoid cortex, but could also be seen scattered individually in the paracortex and cortico–medullary junction. The density of positive-staining cells varied between calves, but there was no
Table 1 Comparison of immunoglobulin-bearing cells in mesenteric lymphoid tissues from group A–D calves
Group A Žfresh colostrum. Group B Žcolostrum-deprived. Group C Neonates Fetuses Group D Žtreated colostrum.
IgG1
IgG2
IgA
IgM
0 2
0 2
0–1 1
3 3
1–2 1 0
1–2 1 0
0–1 1 0–1
3 2 3
The number of cells with positive staining was categorically graded as absent Ž0., low Ž1., medium Ž2. or high Ž3.. A range of density is given where cell numbers varied among individuals in a group.
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recognizable pattern of difference between calf groups. The number of IgM-positive regions per medium powered field and the density of IgM-positive cells per region was always grade 2 to 3 ŽTable 1.. The regions containing IgM-positive cells were equally abundant in mesenteric and peripheral lymph nodes. The density and abundance of IgA-positive cells was also consistent between calves ŽTable 1.. These IgA-positive cells were much less abundant than IgM-positive cells and usually were present as individual cells scattered throughout the cortex, paracortex, and cortico–medullary junction ŽFig. 1.. Occasional aggregates of IgA-positive cells were observed in all groups. IgA-positive cells were very rare in peripheral lymph nodes in any calf group. The major differences between calf groups were confined to the IgG1 and IgG2 positive cells ŽTable 1 and Fig. 1.. In Group A, calves fed fresh colostrum, no IgG1 or IgG2 positive cells were observed in any sections from either mesenteric or peripheral lymph nodes. In contrast, the lymph nodes of colostrum-deprived Group B calves contained numerous IgG1 and IgG2 positive cells, with a similar pattern of distribution and density. The IgG positive cells were frequently present in clusters in cortical regions compatible with the location of lymphoid follicles. Discrete, individual, and small clusters of IgG positive cells were also scattered throughout the cortex, paracortex, and at the cortico–medullary junction. Group C calves were examined to determine what Ig positive cell populations were present prior to colostral ingestion. In the two neonatal Group C calves, IgA and IgM positive cells were present in similar numbers and with a similar distribution to other post-natal calves ŽGroups A, B, and D.. In Group C fetuses, IgA and IgM positive cells were present but in reduced numbers when compared to group A calves. Lymphoid tissues from Group C neonatal calves contained IgG1 and IgG2 positive cells in similar numbers to Group B calves ŽFig. 2.. Similarly, fetuses contained IgG positive cells, although their numbers were markedly reduced. In Group D calves, which received frozen or irradiated colostrum, the numbers and distribution of Ig positive cells in lymphoid tissues were indistinguishable from the Group A calves that received fresh colostrum. 4. Discussion Colostrum is principally regarded as a source of passive immunity in the newborn calf. This study demonstrated that colostrum also has a profound effect on postnatal development of the calf’s endogenous immune system and specifically on the B cell lineage. Examination of mid-term fetuses, neonatal calves, and 36-h old colostrum-deprived calves demonstrated the increasing presence of sIg-positive B cells of all isotypes at each stage. However, ingestion of colostrum changed the number of sIg-positive cells Fig. 2. Immunoglobulin positive cells in the mesenteric lymph nodes of group C newborn calves, 6 h after birth Žmagnification 100=.. Sections are immunoperoxidase stained using mAbs recognizing surface IgG1 Ža., IgG2 Žb., IgA Žc. and IgM Žd.. Numerous IgG1 and IgG2 positive cells are seen throughout the cortex, paracortex and cortico–medullary junction indicating the presence of these cells prior to the ingestion of colostrum. The number and distribution of IgA and IgM cells is similar to those seen in Fig. 1.
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in lymph nodes in an isotype-specific manner resulting in the disappearance of IgG1 and IgG2-positive lymph node cells alone. This observation is consistent with elimination of the IgG-positive B cells that were present at birth, or the downregulation of sIgG expression by these cells. The presence of sIg-positive cells of various isotypes in both fetuses and term calves is consistent with the previously reported ontogeny of antibody production in calves ŽOsburn et al., 1982.. Similarly, the distribution of IgM-positive cells was consistent with the distribution of B-cell areas described in calves and older cattle ŽGaleotti et al., 1993.. The absence of distinct primary and secondary lymphoid follicles and abundance of IgM-positive cells compared to other isotypes may reflect the antigen naivete´ of calves in this study. The correlation of sIg expression with developmental stage has been defined in mice and humans ŽJaneway and Travers, 1997a.. The scattered cortical distribution of IgG and IgA-positive cells seen in fetuses and colostrum-deprived calves in this study is consistent with the description of mature B cells that have recently encountered antigen, interacted with T cells, and undergone antigen-driven class-switching prior to migrating to follicles and establishing germinal centers ŽJaneway and Travers, 1997b.. The presence of IgG-positive and IgA-positive cells in fetuses and colostrum-deprived calves indicates that isotype switching occurs in some B cells before birth. This switch may occur as a result of an in utero exposure to maternal commensal microbes ŽSchultz et al., 1973., or spontaneously, as described for natural or pre-immune antibody production in other neonates ŽCukrowska et al., 1995.. These natural antibodies are produced independent of exposure to environmental antigens, are polyspecific, have low affinity, react with a variety of self-antigens, and are spontaneously formed ŽCukrowska et al., 1996.. The function of natural antibodies is not clear, but they may represent a first line of defense during fetal and neonatal life. In Group D calves, neither a rapid freeze–thaw cycle or g-irradiation prevented colostral ingestion from eliminating sIgG-positive cells in lymph nodes. This finding tends to exclude colostral cells and cold-labile factors as causes of this phenomenon, despite the fact that both of these factors have been shown to affect immunoglobulin production in newborn calves and pigs, respectively ŽRiedel Caspari and Schmidt, 1991; Riedel Caspari, 1993; Klobasa et al., 1990.. Furthermore, changes in IgG-positive cells were not confined to intestinally-associated lymphoid tissues, but were observed in all lymph nodes examined. This indicates that the colostral element was an absorbable, widely distributed, cold-stable factor such as immunoglobulin or a cytokine. Immunoglobulins may be the most likely candidate given their established effect on endogenous antibody production. Colostrum-associated suppression of antibody production correlates with the level of circulating antibody and is isotype-specific ŽHeckert et al., 1991; Husband and Lascelles, 1975; Van Zaane et al., 1986.. In addition, immunoglobulins are cold-resistant, systemically absorbed, and distributed to all tissues ŽBoyd and Boyd, 1987; Haines et al., 1992; Besser, 1993.. A number of potential mechanisms could explain why IgG-positive cells were absent from the lymph nodes of colostrum-fed calves. These include downregulation due to capping and endocytosis of sIg, altered trafficking or the death and removal of sIg-positive cells. Downregulation of sIg expression can occur in the presence of anti-immunoglobulin antibody, and is reversible immediately when the anti-immuno-
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globulin is removed ŽLawton and Cooper, 1979.. This mechanism could explain the findings in this study since colostrum can contain anti-idiotypic antibodies and B cells of the newborn are susceptible to sIg modulation ŽCollins et al., 1991; Raff et al., 1975.. In mice, anti-immunoglobulin antibodies reduce sIg expression in B cells by inducing capping and endocytosis ŽLawton and Cooper, 1979.. In neonatal B cells, this process is irreversible and results in functional inactivation ŽKearney et al., 1978; Raff et al., 1975.. Since colostrum can contain antibodies with extensive anti-immunoglobulin specificities, this mechanism may explain the changes in sIg expression seen in this study ŽCollins et al., 1991.. Passive transfer of antibodies has also been shown to affect B cell populations in the context of intravenous immunoglobulin ŽIVIG. therapy, and can alter an individual’s antibody repertoire by eliminating certain bone marrow B cell lineages or modifying emergent B cell repertoires ŽSunblad, 1994.. It is therefore possible that colostral immunoglobulin changed sIgG-expression by interacting with B cells directly, or by altering T cell function ŽSunblad, 1994.. The cellular mechanisms by which immunoglobulin-induced changes in antibody production are induced are often mediated by cytokines ŽAbe et al., 1994; Andersson et al., 1994; Toyoda et al., 1994.. Alternatively, it remains possible that bovine colostral cytokines ŽSordillo et al., 1991., or other non-immunoglobulin proteins ŽSambasivarao et al., 1996., may have directly down-regulated sIg expression in this study. A major immunological transition occurs at the time of birth, as the neonate is exposed to a large number of foreign antigens in its new environment. The colostrum-induced immunomodulation described in this study may provide a biological advantage during this process. Because colostrum ingestion eliminated only IgG-positive B cells, less mature sIgM-positive cells, and IgA-positive cells in mesenteric lymph nodes with a probable role in mucosal immune responses were spared. This confined the effect to a B cell population most likely to produce IgG antibodies restricted to the circulating and systemic compartments. This may be consistent with suppression of endogenous antibody responses in the calf to circulating passively transferred maternal components, such as immunoglobulins themselves. Overall, this may result in tolerance of maternal immunoglobulins, and an increased longevity of these passively transferred antibodies. Alternatively, suppression of immunoglobulin production in circulating and systemic compartments may also allow the passage of colostral immune cells to the calf. There is indirect evidence that colostral cells upregulate antigen-specific immune responses in the calf, and definitive evidence for the transfer of immune cells between dam and offspring in other species ŽArchambault et al., 1988; Ellis et al., 1996; Le Jan, 1996; Riedel Caspari, 1993; Sheldrake and Husband, 1985; Williams, 1993.. Another potential immunomodulatory role for colostral antibodies may be influencing the calf antibody repertoire through idiotyperanti-idiotype interactions ŽKeller et al., 1991; Wikler, 1980.. This effect has been demonstrated in mice where neonates receiving antibodies bearing certain idiotypes were protected against infection, and were also primed for higher antigen-specific antibody responses on subsequent immunizations ŽOkamoto, 1991.. It is possible that the elimination of IgG-positive cells may result from such idiotyperanti-idiotype interactions. While any advantage of the elimination of IgG-positive B cells in calf lymph nodes subsequent to colostral ingestion remains uncertain, this is a further example of the
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immunomodulatory effects of colostrum in calves. An increased understanding of such interactions will have important implications for our understanding of the full immunological role of colostrum, and for our capacity to influence calf immune responses by active or passive vaccination.
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