The interaction of infant formula with macrophages: effect on phagocytic activity, relationship to expression of class II MHC antigen and survival of orally administered macrophages in the neonatal gut.

The effect of infant formula on human peritoneal and breast milk macrophages has been investigated. The ability of peritoneal macrophages to subsequently ingest and degrade immune complexes was slightly impaired, but breast milk cells were not affected. However, the cells were found to have bound antigenically intact casein and beta-lactoglobulin, although little, if any, alpha-lactalbumin was bound. Furthermore, a positive correlation was found between binding of these proteins and expression of HLA-DR antigen. Labelled macrophages fed to newborn mice survived for at least 4 hr in the gastrointestinal tract and, in some cases, localized in the mucosal tissue. In one case a labelled cell was found in the spleen. These findings indicate that breast milk macrophages may be able to perform immunological functions in the gut, and suggest that binding of cows' milk proteins by macrophages may constitute a first step in the sensitization of the neonate to cow's milk proteins. Human milk macrophages may also play a protective role by acting as antigen-presenting cells in the local immune response of the gut.

Suppressor T cells, antigen-presenting cells and the role of I-J restriction in oral tolerance to ovalbumin.

Suppressor T cells (Ts) and antigen-presenting cell (APC) activity are both important for the induction of systemic tolerance after feeding protein antigens to mice. In this report, we have examined further the nature of the inter-relationship between Ts and APC in oral tolerance to ovalbumin (OVA). We found previously that oral tolerance to OVA could prevented by treating mice with oestradiol, and we now report that oestradiol enhances the ability of spleen APC to present OVA to T cells. In parallel, mice treated with oestradiol do not generate the Ts activity normally found after feeding OVA. Treatment of mice with anti-I-J antiserum prevents the induction of both tolerance and Ts activity after feeding OVA, but the suppressor effector cells generated by feeding OVA can not be depleted in vitro by treatment with anti-I-J antibody plus complement. In vivo administration of monoclonal anti-I-A antibody had no effect on oral tolerance to OVA. Our results show that induction of oral tolerance to OVA is an I-J-restricted phenomenon and we propose that this reflects an interaction between specific Ts cells and a population of I-J+ cells which we suggest are APC.

Functional characteristics of intraepithelial lymphocytes from mouse small intestine. III. Inability of intraepithelial lymphocytes to induce a systemic graft-versus-host reaction is because of failure to migrate in vivo.

In this study we have investigated whether addition of bone marrow accessory cells or concurrent administration of recombinant IL-2 would allow intraepithelial lymphocytes (IEL) to induce a systemic, lethal GvHR in irradiated hosts. In addition we have studied the ability of IEL to migrate into lymphoid tissues after intravenous injection and compared this with their locomotor capacity in vitro.

Modulation of in vitro thymidine incorporation into crypt cells from the murine small intestine.

A method is described for the isolation of enriched populations of crypt cells from the murine small intestine. The method was developed to study the response of cells to various stimuli in vitro. The properties of the isolated cell preparations varied with the state of the intestinal mucosa of the mice from which they were isolated. Thus we could distinguish between cells from lactating and non-lactating mice. Polyamines, which are putative modulators of crypt cell division, failed to stimulate [3H]TdR incorporation in vitro. Lymphocyte culture supernatants suppressed [3H]TdR incorporation at dilutions of 1:4 to 1:64. Supernatants of 12-O-tetradecanoylphorbol-13-acetate-stimulated EL-4 cells and of mixed lymphocyte cultures failed to stimulate [3H]TdR incorporation of any dilution. Supernatants of concanavalin A-stimulated spleen cells gave less suppression of [3H]TdR incorporation than those of unstimulated spleen cells and stimulated incorporation at dilutions of 1:64 and 1:128. Phytohaemagglutinin stimulated [3H]TdR incorporation at high concentrations, whereas concanavalin A (con A) had no effect. This study shows that the isolated murine crypt cells may have the potential to provide a useful in vitro model for crypt cell responses to stimuli.

The incidence, clonal origin and secretory nature of serum paraproteins in chronic lymphocytic leukaemia.

Immuno-isoelectric focusing (IIEF) showed a 61% incidence of serum paraproteinaemia in 56 patients with chronic lymphocytic leukaemia (CLL). A strong correlation between the serum paraprotein heavy chain isotypes and those of the cytoplasmic heavy chain immunoglobulins was observed with no discrepancy noted in light chain expression. Density gradient ultracentrifugation analysis of selected sera containing monoclonal IgM showed that the IgM paraproteins were mostly 19S, secretory IgM but one patient was found to have both 19S and 8S monoclonal IgM. When the cellular origin of the IgM and IgD paraproteins found in one patient was investigated, both paraproteins were found to share the same idiotype and originate from the neoplastic clone. These findings confirm the view that there is an incomplete maturation block in chronic lymphocytic leukaemia and that in vivo secretion of paraproteins by the neoplastic cells is a relatively common occurrence.

The incidence of monoclonal gammopathy in a population over 45 years old determined by isoelectric focusing.

Immuno-isoelectric focusing (IIEF), a technique previously shown to be sensitive for the detection of paraproteinaemia, was used to test 200 individuals over the age of 45, without history of B cell neoplasm, for the presence of serum paraproteinaemia. 11% of these individuals had evidence of paraproteinaemia detectable by IIEF compared with only 2% by zonal and immunoelectrophoresis. A further 12% had oligoclonal immunoglobulins and the remainder had no qualitative abnormality of the immunoglobulin profile. These results are discussed with particular reference to the aetiology, diagnosis and monitoring of potential B cell neoplasm in high risk individuals or groups.

Quantitation of monoclonal immunoglobulins by immuno-isoelectric focusing and its application for monitoring secretory B cell neoplasia.

A scheme for quantitation of serum and urinary paraproteins is described using isoelectric focusing and scanning densitometry. Paraproteins could be quantified using this system when present at concentrations ranging from 1 mg/ml to 26 mg/ml, depending on the immunoglobulin class. The relevance of these results to monitoring secretory B cell neoplasia and the distinction between monoclonal gammopathy of undetermined significance and myeloma is discussed.

Natural killer cell activity in atopic dermatitis: a sequential study.

It is well-recognized that patients with atopic dermatitis handle certain cutaneous viral infections poorly. As natural killer (NK) cell activity is considered to contribute to the immune response to viral infection, seven young adults with atopic dermatitis had their NK cell function assessed over a 12-month period. Natural killer cell activity was found to correlate inversely with disease activity. The more active the disease, the greater was the reduction in NK cell function (P less than 0.01. In addition, a strong correlation between clinical activity and IgE was shown (P less than 0.001).

Effect of human colostrum and infant formula on the phagocytic activity of macrophages. I. Resident and stimulated mouse peritoneal macrophages.

Phagocytosis and degradation of radiolabelled human transferrin-anti-transferrin immune complexes by resident and stimulated mouse peritoneal macrophages was inhibited by liquid infant formula, particularly in the case of resident cells. Mouse peritoneal macrophages exposed to infant formula were shown by immunofluorescence to bind casein and beta-lactoglobulin, but there was little binding of alpha-lactalbumin. Comparison of various artificial milks, cow's milk and purified casein indicated that both the concentration and the degree of denaturation of casein may be important in the impairment of macrophage function by milk. It is suggested that bottle feeding of infants might result in impairment of macrophage function in the small intestine.

Appearance of delayed-type hypersensitivity effector cells in murine gut mucosa.

Feeding of a protein antigen, human gamma globulin (HGG), to BALB/c mice prior to parenteral immunization resulted in the abrogation of a delayed-type hypersensitivity (DTH) response to challenge with that antigen. Unlike parenterally immunized mice, HGG-fed mice were unable to transfer DTH to naive syngeneic recipients using peripheral lymph node lymphocytes. Co-transfer experiments ruled out the possibility of a suppressor cell in the orally immunized mice operating on DTH effector cells. Intra-epithelial lymphocytes (IELs) from mice immunized either orally or parenterally were able to transfer a DTH reaction to unimmunized recipients, while mesenteric lymph node lymphocytes from orally, but not parenterally, immunized donors were capable of transferring DTH. The implications of these results for investigations of gastrointestinal disorders with a suspected immunological aetiology are discussed.

Natural-killer-cell activity in atopic dermatitis.

Natural-killer (NK)-cell activity was measured in the peripheral blood of twenty patients with atopic dermatitis and in a group of thirteen age-matched non-atopic controls (nine subjects on thirteen occasions). The method uses a chromium-release assay with the human leukaemia cell line, K562, labelled with 51Cr as the target cell. A highly significant reduction in NK-cell activity was found in the patients with atopic dermatitis.

Serum paraproteins in chronic lymphocytic leukaemia.

The presence of paraproteins in the sera of 10 patients with chronic lymphocytic leukaemia (CLL) was investigated using immunoisoelectric focusing. Monoclonal immunoglobulins were found in nine of these 10 sera. Five sera contained a single monoclonal IgM paraprotein, one serum contained a single monoclonal IgG paraprotein, while three sera contained more than one monoclonal paraprotein--namely, IgM + IgD, IgM + IgG, and IgM + IgD + IgG. The results indicate that the malignant B cells of CLL may be at a later stage of differentiation than previously assumed and serum monoclonal immunoglobulin could be of value as a tumour marker.

Augmentation of intestinal and peripheral natural killer cell activity during the graft-versus-host reaction in mice.

We have investigated the possibility that nonspecific cytotoxicity may be involved in the pathogenesis of the intestinal phase of the graft-versus-host reaction (GVHR) in mice. A GVHR was induced in unirradiated (CBA X BALB/c)F1 mice and natural killer (NK) cell activity against YAC-1 followed in the spleen, mesenteric lymph node (MLN), and isolated intraepithelial lymphocytes (IEL). Augmented NK activity developed simultaneously in all tissues in parallel with the progress of the GVHR. The NK activity of IEL also showed a close association with the increased numbers of IEL found on sections of small intestine. Mature T lymphocytes and macrophages did not contribute to the nonspecific cytotoxicity, and antihost cytotoxic T cells were not detected in any tissue. The results indicate that generalized recruitment of NK cells occurs during the GVHR both in peripheral and intestinal lymphoid tissues, and we propose that lymphokines are responsible for this phenomenon. NK cells recruited by a delayed-type hypersensitivity reaction may contribute to the pathogenesis of the GVHR, but an alternative explanation is that NK cells may inhibit the progression of the GVHR.

Analysis of the effector functions of different populations of mucosal lymphocytes.

Lymphocytes separated from the epithelial layer of mouse small intestine, IEL, were tested for their NK cytotoxicity against Yac-1 targets. There was little NK activity in a 4 hour assay, but high activity in an 18 hour assay, and the NK activity of IEL did not parallel that in the spleen in any of the mouse strains tested. Furthermore, IEL exerted a suppressor activity on mouse spleen NK activity. Specific T-cell cytotoxicity appeared in IEL in mice immunized with an intraperitoneal injection of P-815 tumor cells. By contrast with IEL, LPL had little NK or NK suppressor activity, but higher levels of specific T-cell cytotoxicity in tumor-immunized mice than intraepithelial lymphocytes. A high proportion of IEL had granules that stained with Giemsa and Astra blue. Furthermore many IEL carried Lyt-2+ phenotype and no other T-cell surface antigen. Intraepithelial lymphocytes appeared, therefore, to have staining and phenotype characteristics of both granular NK cells and suppressor cells. It was clear that the intestinal mucosa contained populations of immune effector cells that were heterogeneous in nature and function.

Analysis of natural killer effector and suppressor activity by intraepithelial lymphocytes from mouse small intestine.

Intraepithelial lymphocytes (IEL) are morphologically similar to NK cells in other tissues and we have studied the NK activity of IEL isolated from mouse small intestine. In contrast to spleen NK cells, IEL showed little activity against YAC-1 over 4 h but had high levels of NK activity when the assay was extended to 18 h. IEL from nude mice did not show the enhanced NK activity found in other tissues. IEL were also found to suppress the NK activity of spleen cells and this suppressor function was not mediated by T lymphocytes or macrophages. The results indicate that the intestinal epithelium contains a population of potent NK cells which may represent a type of NK cell different to that found in other tissues. In addition, there are also cells capable of regulating NK cell function in the epithelial layer.

Cytotoxic T cells in small intestine epithelial, lamina propria and lung lymphocytes.

We have examined the development of specific cytotoxic T-cell activity in the lungs and the epithelium and lamina propria of the small intestine following tumour cell inoculation by subcutaneous or intraperitoneal routes. After an intraperitoneal injection of tumour cells, large amounts of cytotoxic activity are detectable in the lungs and lamina propria. In comparison, the epithelial lymphocytes of the small intestine display low cytotoxic activity. After a subcutaneous injection, little cytotoxicity is detectable except in the lungs and the development of such cytotoxicity has a much shorter time course compared with that induced by an intraperitoneal inoculation of tumour cells. The data indicate a marked difference in the functional capacity of lymphocytes from the epithelium and lamina propria of the small intestine.

Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine.

Existing methods for the production of lymphocytes from the small intestine have proved unsatisfactory when applied to the mouse. We report here a new method for the production of highly pure suspensions of lymphoid cells from the epithelial layer and lamina propria of mouse small intestine. The production and purification methods are described in detail. At least ten million lymphocytes are obtainable from each small intestine from either the epithelium or lamina propria and the cell suspensions are shown to be little contaminated by non-lymphoid cells. Preliminary analysis of the two cell types indicates that they belong either to two separate populations or to one population in very different stages of differentiation. The use of purified lymphoid cells from the epithelium and lamina propria of the small intestine may enable examination of the generation of cytotoxicity towards gut epithelial cells; this may be important in the development of inflammatory bowel diseases.

The early appearance of specific cytotoxic T cells in murine gut mucosa.

Using a new technique for isolating lymphoid cells from the lamina propria of murine small intestine, we have examined the appearance of specific cytotoxic T cells in the gut following intraperitoneal immunization with an allogenic tumour. Specific cytotoxic T cells appeared in the lamina propria at a time when there are very few cytotoxic lymphocytes in any of the organized lymphoid tissues. Greater levels of cytotoxicity were found in the gut compared with any other site for at least 3 weeks following a single injection of tumour cells.