Dennis L. Watson

Immunomodulatory effects of dietary whey proteins in mice

Studies on the immunomodulatory properties of dietary whey proteins in mice are reported. Ingestion of bovine milk whey proteins, either as a supplement in an adequately balanced commercial diet or as the only protein source in a balanced diet, consistently enhanced secondary humoral antibody responses following systemic immunization with ovalbumin, when compared with other protein sources such as soyabean protein isolate and ovine colostral whey proteins. After 5-8 weeks of feeding, dietary milk whey proteins enhanced cell-mediated immune responses as revealed by footpad delayed-type hypersensitivity responses, and concanavalin A-induced spleen cell proliferative responses. To monitor nutritional effects of milk whey proteins, live weight, leucocyte counts and clinical changes of diet-fed mice were examined. The present results confirm other previous results that dietary bovine milk whey proteins have immunoenhancing properties in mice and these properties are unlikely to be related solely to the nutritional effects.

Effects of purified bovine whey factors on cellular immune functions in ruminants

The immunomodulatory properties of bovine milk and whey have long been documented. The recent advance of whey protein fractionation technology has now allowed us to study the immunobiological properties of some highly purified components of whey, with a view to exploiting their possible industrial and biomedical applications. The effects of fractionated bovine whey proteins on cellular immune responses were therefore examined using a panel of in vitro assays. Both lactoferrin (LF) and lactoperoxidase (LP) were found to inhibit proliferation and interferon-gamma (IFN-gamma) production of ovine blood lymphocytes in response to mitogenic stimulation. However, their effects in a combined fraction or in whey protein concentrate (WPC) were either diminished or eliminated. LF and LP had no effect on lipopolysaccharide (LPS)-induced ovine blood lymphocyte proliferation, production of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF alpha) by ovine bronchoalveolar lavage (BAL) macrophages, major histocompatibility complex (MHC) Class II antigen expression by ovine BAL macrophages and bovine natural killer (NK) cell activity. However, alpha-lactalbumin (alpha LA) exhibited an enhancing effect on IL-1 beta production. It is noteworthy that as bovine whey fractions become progressively more purified, their modulatory effects on the immune response also become more clear-cut. The effects of LF, LP and alpha LA may be eliminated by their combination in whey or by other minor components of whey. Further investigation of industrial applications for whey proteins of high purity is warranted.

Effect of iscoms and their adjuvant moiety (matrix) on the initial proliferation and IL-2 responses: comparison of spleen cells from mice inoculated with iscoms and/or matrix.

In the iscom, antigen is attached by hydrophobic interactions to a matrix which is built up by the adjuvant Quil A and lipids. Thus, the iscom presents antigen in multiple copies on a small particle with a built-in adjuvant. By studying the specific antibody response, in vitro proliferation and IL-2 secretion by splenocytes from mice following different in vivo treatments with iscoms and/or matrix, we attempted to distinguish between nonspecific stimulatory effects, caused by the matrix or iscoms, and specific responses to the antigens incorporated into iscoms. The results strongly suggest that matrix and also iscoms exert a nonspecific adjuvant activity by a transient high spontaneous proliferation of cells collected within 2 weeks after administration of iscoms or matrix. This high rate of proliferation was preceded by suppressed proliferation, 3 days after injection with matrix or iscom. The adjuvant component included in iscoms, i.e., the matrix, does not excert a mitogenic stimulation in vitro or influence the levels of specific antibodies in serum. Specific responses to the antigens included in iscoms were recorded both as increasing levels of serum antibodies and as iscom-induced proliferation of immune spleen cells in vitro. The recruitment of IL-2 was only related to the specific stimulation induced by the antigens in iscom.

Interactions between Immune‐Stimulating Complexes (ISCOMs) and Peritoneal Mononuclear Leucocytes

Studies were undertaken in mice using immune‐stimulating complexes (ISCOMs) or micelles prepared from envelope glycoproteins of human influenza virus (PR8) and matrix (i.e., ISCOM skeleton without incorporated antigen). Electron microscopic studies showed that ISCOMs, in contrast to micelles, have a remarkable affinity for cell membranes and seem to rapidly promote their own internalization by cells to which they adhere. PR8 ISCOMs, but not matrix nor micelles, significantly increased the expression of membrane Ia by peritoneal mononuclear leucocytes 24 hr after intraperitoneal immunization.

Modulation of immune responses by bovine |[beta]|-casein

The present study reports the influence of bovine β‐casein on in vitro and in vivo immune responses. Bovine β‐casein showed an inhibitory effect on ovine neutrophil chemotaxis but had an enhancing effect on superoxide production by neutrophils. In response to mitogenic stimulation, the proliferative response of both T and B lymphocytes was significantly enhanced by β‐casein. While β‐casein had no significant effects on IFNγ production by ovine blood lymphocytes, and TNFα production and MHC Class II antigen expression by ovine bronchoalveolar macrophages, it enhanced IL‐1β production by the macrophages. β‐casein also had no influence on bovine NK cell activity against a virally‐infected cell line. Interestingly, β‐casein was found to reduce the adjuvant effect of matrix immune stimulating complexes (ISCOM) on anti‐ovalbumin antibody response in mice when given intramuscularly. Taken together, the results suggest that bovine β‐casein had selective modulating effects in vitro on both innate and adaptive immune responses in ruminants, whereas systemic administration of β‐casein, that might have a depressive effect on adjuvant activity, requires further study.

Influence of whey and purified whey proteins on neutrophil functions in sheep

The effects of ruminant whey and its purified fractions on neutrophil chemotaxis and superoxide production in sheep were studied. Both colostral whey and milk whey were found to inhibit chemotaxis regardless of whether they were autologous or homologous, but the inhibitory effects were abolished by washing neutrophils with culture medium before their use in the chemotaxis assay. Colostral whey and milk whey also inhibited the chemotactic activity of zymosan-activated serum. Whey fractions of various degrees of purity such as lactoferrin, lactoperoxidase, lactoferrin-lactoperoxidase, alpha-lactalbumin, bovine serum albumin and whey protein concentrate were then studied. While none of these proteins showed any effects on chemotaxis, lactoferrin-lactoperoxidase and whey protein concentrate were found to have an enhancing effect on superoxide production in a dose-dependent manner. Our results provide information on the modulatory role of ruminant milk proteins in inflammatory responses and warrant future investigation.

Immunomodulatory activities of whey fractions in efferent prefemoral lymph of sheep.

Studies on the immunomodulatory activities of ruminant milk and colostral whey fractions were undertaken. By comparing with boiled colostral whey in a preliminary experiment, a putative heat-labile immunostimulatory factor for antibody responses was found to be present in ovine colostral whey. Studies were then undertaken in sheep in which the efferent prefemoral lymphatic ducts were cannulated bilaterally, and immune responses in the node were measured following subcutaneous injection in the flank fold of whey protein preparations of various purities. A significant sustained decline of efferent lymphocyte output was observed following injection with autologous crude milk whey or colostral whey preparations, but no changes were observed in interferon-gamma levels in lymph plasma. Two bovine milk whey fractions (lactoperoxidase and lactoferrin) of high purity were compared in bilaterally cannulated sheep. A transient decline over the first 6 h was seen in the efferent lymphocyte output and lymph flow rate after injection of both fractions. A significant difference was seen between the two fractions in interferon-gamma levels in lymph at 6 h after injection. However, no significant changes in the proportion of the various efferent lymphocyte phenotypes were seen following either treatment. Whereas both fractions showed a significant inhibitory effect in a dose-dependent manner on the proliferative response of T lymphocytes, but not B lymphocytes, to mitogenic stimulation in vitro, no similar changes were seen following in vivo stimulation with these two fractions.

Factors in ruminant colostrum that influence cell growth and murine IgE antibody responses

Bovine colostrum was investigated as a source of biologically active molecules capable of stimulating the growth of mammalian cells in culture and modifying the immune response in a murine model. An extract prepared from bovine colostral whey by cation exchange and reversed-phase chromatography stimulated the growth of L6 rat myoblasts, Balb/c-3T3 mouse fibroblasts and BHK-21 baby hamster kidney cells with equal or greater potency than fetal bovine serum. Fractionation of the bovine colostral extract by gel-permeation chromatography in M-acetic acid identified a number of cell-growth factors for each cell type. Bovine colostral extract was compared with an ovine colostral whey preparation for its ability to modulate IgE antibody responses in mice. Doses of 8 and 4 mg/d of ovine colostral whey or bovine colostral extract specifically suppressed IgE antibody responses, whereas at lower doses suppression did not occur. We conclude that bovine colostrum contains cell-growth factors as well as immunomodulatory factors that are able to regulate the IgE response in a heterologous species.

Effect of dextran sulphate on IgG subclass of antibody in efferent popliteal lymph of sheep

IgG1 and lgG2 participation in anti‐hapten, anti‐carrier and immunoglobulin‐containing cell (Ig‐cc) responses was studied in sheep immunised with killed Staphylococcus aureus‐Dinitrophenyl (DNP) conjugates. The antigen was given with or without the polyanionic adjuvant dextran sulphate (DXS). Animals were immunised intracutaneously on the lateral hock of one hind leg with 1 × 109 S. aureus‐DNP with or without 20 mg DXS. Six weeks later, primed sheep underwent surgery to cannulate an efferent popliteal lymphatic vessel in the immunised leg. Forty‐eight hours after surgery, each sheep was given a further intracutaneous injection of 1 × 109 S. aureus‐DNP with or without 20 mg DXS adjacent to the primary injection site. IgG1‐cc and IgG2‐cc in lymph were counted using indirect immunofluorescence. Total IgG1 and IgG2 anti‐staphylococcal and anti‐DNP antibody output in lymph was determined using enzyme‐linked immunosorbent assays (ELISAs). DXS markedly increased antibody responses to S. aureus‐DNP in the popliteal lymph node of sheep and was shown to strongly enhance immunological memory. The ratios of IgG2‐cc:IgG1‐cc and lgG2:IgG1 anti‐staphylococcal antibody in lymph‐draining popliteal lymph nodes of sheep stimulated with DXS and S. aureus‐DNP was significantly greater than the same ratios in animals given antigen alone. The IgG subclass‐specific immunomodulatory effects of DXS were exerted whether the adjuvant was given with primary and/or secondary inoculations. There was no difference in the ratio of IgG2:IgG1 anti‐DNP antibody in lymph from animals given S. aureus‐DNP with and without DXS.

Comparison of immunomodulatory properties of purified lactoferrin, lactoperoxidase and β-casein in sheep

Bovine milk contains a variety of proteins and peptides that are biologically active (Ogra & Ogra, 1978; Duncan & McArthur, 1981; Newby et al . 1982; Juto, 1985; Stoeck et al . 1989; Mincheva-Nilsson et al . 1990; Watson, 1990; Barta et al . 1991; Politis et al . 1991; Fiat et al . 1993). Our laboratory has a long-term interest in some purified milk proteins, particularly lactoferrin (LF), lactoperoxidase (LP) and β-casein (β-CN), which have been shown to be immunologically significant. Some of our recent studies on these bovine milk proteins, particularly β-CN, indicated that their in vitro immunological effects did not always parallel their in vivo activities (Wong et al . 1996 a , b ; 1997 a , b ). This study was designed to investigate and compare the capacity of these purified bovine milk proteins to modulate a range of components that are vital to in vivo immune responses in sheep, with a view to providing further information on their potential in biomedical applications. To achieve this objective, a sensitive lymphatic cannulation model was employed that allows in vivo immune components and their functions to be measured in lymph collected under physiological conditions.