The effect of topical steroid application on natural killer cell activity.

Peripheral blood natural killer (NK) cell activity of a group of 10 healthy non-atopic volunteers was reduced following the topical application of 15 g of 0.1% betamethasone valerate ointment to the skin nightly for 1 week. In contrast, no such effect was observed when the inactive base of the steroid ointment was used. NK cell activity dropped significantly by day 7 (P less than 0.05) and then recovered, although NK cell activity at day 22 was still lower than that observed at the start of the experiment. These findings suggest that topically applied steroid is absorbed in sufficient amounts to cause a systemic effect on NK cell function. This may have implications in a number of dermatological disorders, including atopic dermatitis, where topical steroids form the mainstay of treatment.

Investigation of the immune status of mice during and following selective decontamination of the digestive tract.

Selective decontamination of the digestive tract (SDD) employs oral antibiotics to eliminate aerobic Gram-negative bacilli while retaining the anaerobic flora. A combination of SDD and parenteral cefotaxime has recently been reported to strikingly reduce the incidence of infection in patients treated in an intensive therapy unit. The present study describes the effects of SDD and of cefotaxime on the immune response of mice to protein antigens. The in vivo cellular response to ovalbumin and sheep red blood cells was unchanged. However, SDD appeared to decrease the in vitro mitogenic response of spleen cells to phytohaemagglutinin, and cefotaxime similarly affected the response to Concanavalin A. The antibody response to sheep red blood cells was increased in the period after discontinuation of SDD. The antibody response was otherwise not affected. These results indicate that SDD is unlikely to have adverse effects on the immune response to protein antigens.

Priming of systemic and local delayed-type hypersensitivity responses by feeding low doses of ovalbumin to mice.

We have examined the effects on both systemic and intestinal immunity of feeding different doses of ovalbumin (OVA) to mice. A single feed of doses of more than 1 mg OVA produced significant suppression of subsequent delayed-type hypersensitivity (DTH) and IgG antibody responses. Feeding 100 micrograms-1 mg OVA had no net effect on systemic immunity, but mice fed 10-50 micrograms OVA had consistently enhanced systemic DTH responses when immunized subsequently with OVA in adjuvant. Oral challenge of these mice with OVA produced alterations in mucosal architecture and in intra-epithelial lymphocyte counts, consistent with the presence of an intestinal DTH response. Similar changes were not found in mice fed tolerogenic doses of OVA. Although feeding low doses of OVA primed both systemic and intestinal DTH responses, this had no effect on serum IgG responses and very little systemic DTH could be revealed in OVA-fed mice without systemic challenge with OVA in adjuvant. We conclude that feeding certain low doses of protein antigens can induce priming of local and systemic DTH responses rather than the immune tolerance which is normally found. The development of clinical food hypersensitivities may be highly dependent on the dose of dietary antigen at the time of first encounter.

Functional properties of intra-epithelial lymphocytes from mouse small intestine. IV. Investigation of the proliferative capacity of IEL using phorbol ester and calcium ionophore.

Intra-epithelial lymphocytes (IEL) from mouse small intestine normally show very poor proliferative responses to mitogens or antigens in vitro. In this report, we show that purified IEL give a moderate proliferative response when stimulated with a combination of phorbol myristate acetate (PMA) and the calcium ionophore, A23187. Nevertheless, the response of IEL to these agents was much less than that of other lymphoid cells, even when stimulated additionally with concanavalin A (Con A). Furthermore, in comparison with spleen cells, the optimal response of IEL to PMA required higher concentrations of A23187. Optimally stimulated IEL made significant amounts of T-cell growth factor and addition of rIL-2 to the cultures did not enhance the optimal response of IEL to PMA + A23187. Subsequent studies showed that the majority of IEL which respond to PMA + A23187 are L3T4+ Lyt-2-, despite the fact that Lyt-2+ cells from the spleen and thymus responded well to these agents. Our study indicates that the majority of IEL have a very low proliferative potential in vitro and this may reflect a large population of IL-2-unresponsive Lyt-2+ IEL.

Immunological studies of NK cell-deficient beige mice. II. Analysis of T-lymphocyte functions in beige mice.

Lymphocytes from natural killer (NK) cell-deficient beige mice have a poor ability to induce many different forms of graft-versus-host reaction (GvHR). In this study, we have examined whether this defect could reflect an associated abnormality in beige T-lymphocyte function. Compared with normal, congenic C57Bl/6 (B6) mice, beige mice had similar numbers and proportions of T-cell subsets and generated normal allospecific delayed-type hypersensitivity (DTH) responses in vivo. In addition, beige mice developed normal or enhanced DTH responses to the protein antigen, ovalbumin (OVA), and were fully susceptible to the induction of tolerance by feeding OVA. Beige lymphocytes recirculated normally in vivo and showed enhanced proliferative responses to mitogens and alloantigens in vitro. In contrast, beige responder cells generated poor cytotoxic T-lymphocyte (CTL) responses in vitro and had quantitatively identical defects in CTL and NK cell activation after alloimmunization in vivo. These results suggest that although many effector and regulatory T-cell functions are normal in beige mice, the NK-cell defect in these animals is paralleled by impaired CTL activity. We suggest that abnormal T-cell function accounts for the inability of beige lymphocytes to induce GvHR.

Experimental studies of immunologically mediated enteropathy. Development of cell mediated immunity and intestinal pathology during a graft-versus-host reaction in irradiated mice.

The intestinal component of a graft-versus-host reaction (GvHR) provides a useful experimental model to elucidate the pathogenesis of clinical enteropathies which cause villus atrophy and crypt hyperplasia and which are associated with a local immune response. One to three days after induction of GvHR in heavily irradiated (CBAxBALB/c)F1 mice, a proliferative form of enteropathy developed. Compared with controls, these mice had increased counts of jejunal intraepithelial lymphocytes and had a four-fold increase in crypt cell production rate as well as an increase in crypt length. These changes were accompanied by a marked enhancement of splenic natural killer cell activity. After day three, the crypt cell production rate fell to zero and cytotoxic T lymphocytes (CTL) which could lyse targets of host origin appeared. In parallel, mice with GvHR developed significant villus shortening and their clinical condition deteriorated. Further experiments showed that increased counts of intraepithelial lymphocytes, villus atrophy and crypt hyperplasia also occurred in grafts of fetal CBA intestine implanted under the kidney capsule of (CBAxBALB/c)F1 mice with GvHR. As these grafts are syngeneic to the injected CBA spleen cells, they should not be attacked by anti-host cytotoxic T lymphocytes. We suggest that the proliferative and destructive components of enteropathy in GvHR are caused by lymphokines released by an anti-host delayed type hypersensitivity reaction.

The effect of random blood transfusions on immunoglobulin production by peripheral blood mononuclear cells from uraemic patients.

The effect of blood transfusion on humoral immunity in chronic renal failure was studied by examining immunoglobulin production in vitro, in patients awaiting renal transplantation. Pokeweed mitogen (PWM) induced IgG plaque formation was normal in non-transfused uraemic patients while both spontaneous and Staphylococcus aureus Cowan I (SAC) induced immunoglobulin production were reduced. Five to ten units of third party blood transfusion reduced PWM-driven B cell differentiation, but had no effect on SAC-induced plaque formation, while spontaneous production of immunoglobulin was either enhanced or unaffected. As it is known that the response to SAC is less affected by suppressor T cell activity than that to PWM, these differences in the inhibitory effects of blood transfusion on B cell differentiation are further evidence that transfusion may act by increasing suppressor T cell activity.

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.

Genetic control of oral tolerance to ovalbumin in mice.

We have investigated the genetic basis of oral tolerance to OVA in a number of inbred mouse strains. Our results emphasise the efficiency of the oral route for inducing tolerance and provide evidence for both MHC and non-MHC linked control of oral tolerance.

In vitro analysis of B lymphocyte function in uraemia.

We have investigated the immune responses in vitro of uraemic patients undergoing regular haemodialysis or continuous ambulatory peritoneal dialysis. Twenty-five healthy subjects were also studied as controls. In uraemic patients, the number of T and B lymphocytes were within the normal range, but proliferative responses to phytohaemagglutinin (PHA) were impaired. Spontaneous immunoglobulin plaque forming cell (PFC) responses by peripheral blood mononuclear cells (PBMC) from uraemic patients were significantly lower than those of healthy subjects. The PFC response of uraemic PBMC to the T cell independent polyclonal B cell activator (PBA) Epstein-Barr virus (EBV) was comparable to the response of the healthy subjects, indicating that uraemic B cells are still capable of synthesizing immunoglobulin. Pokeweed mitogen (PWM) induced PFC responses of uraemic PBMC were also normal, whereas the response to another T cell dependent B cell activator, Staphylococcus aureus Cowan I (SAC), was very low. Addition of indomethacin to PWM- and SAC-activated cultures of uraemic PBMC enhanced the PFC response to SAC, but had little effect on the PWM response. As full differentiation of B cells in response to SAC depends on helper T cells, we conclude that a defect in T lymphocyte function accounts for the reduced spontaneous and SAC induced production of immunoglobulin by uraemic PBMC. This defect may be mediated by an indomethacin-sensitive mechanism.

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.

The effect of selective decontamination of the digestive tract with the addition of systemic cefotaxime on the aerobic faecal flora of mice.

The administration per-orally to mice of the non-absorbable antibiotics polymyxin E, tobramycin and amphotericin B resulted in the elimination of detectable aerobic gram-negative rods from the faecal flora without affecting the total viable aerobic count. The addition of parental cefotaxime to the regime caused a fall in the number of aerobic lactobacilli and an increase in the number of enterococci. The rise was associated with the translocation of viable enterococci to the mesenteric lymph nodes and the spleen. The changes induced by cefotaxime were reversed when the antibiotic was withdrawn. Following withdrawal of all antibiotics the total aerobic faecal flora increased to above normal levels, but there was no associated diarrhoea. Attempts to implant exogenous enterobacteria into the digestive tract resulted in only low level colonization both in treated mice and in control mice. These results may have implications for the use of this antibiotic regime for selective decontamination of the digestive tract in humans, particularly those who are immunocompromised.

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 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.

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.

Divergent effects of bacterial lipopolysaccharide on immunity to orally administered protein and particulate antigens in mice.

We have investigated whether bacterial lipopolysaccharide (LPS) influences immune responses to dietary protein antigens in experimental animals. Simultaneous intravenous administration of LPS to normal mice fed ovalbumin (OVA) prevented the induction of tolerance for serum IgG antibody responses but did not alter the tolerance of systemic delayed-type hypersensitivity (DTH). In addition, exogenous LPS did not enhance the ability of spleen accessory cells to present OVA to primed T cells. LPS-unresponsive C3H/HeJ mice developed full tolerance of both humoral and cell-mediated immunity after feeding a range of doses of OVA that was equal in degree and persistence to that seen in normal, congenic C3H/HeOla mice and also had normal antigen-presenting cell (APC) activity for OVA. In contrast, C3H/HeJ mice were primed by feeding SRBC instead of developing the systemic tolerance found in normal C3H mice. Our results indicate the complexity of mechanisms that may regulate systemic immunity to orally administered antigens of different forms. Nevertheless, LPS does not modulate DTH responses to fed OVA and does not enhance APC activity, and we conclude that bacterial LPS may be unable to influence hypersensitivity to dietary proteins in man.

Functional characteristics of intraepithelial lymphocytes from mouse small intestine. II. In vivo and in vitro responses of intraepithelial lymphocytes to mitogenic and allogeneic stimuli.

Although isolated intraepithelial lymphocytes (IEL) have been shown to have specific and non-specific cytolytic functions, their ability to proliferate in response to T-cell mitogens or alloantigens is controversial. Here we show that IEL from mouse small intestine do not respond to mitogens such as concanavalin A and phytohaemagglutinin A or in mixed lymphocyte reactions unless an accessory spleen cell is also present. Adherent spleen cells possess the most potent helper function, but a dividing accessory cell may also be required. Supernatants from stimulated lymphocytes also assist IEL to proliferate in vitro, particularly in the presence of adherent accessory cells. IEL could not mediate lethal graft-versus-host disease in irradiated hosts, but could produce popliteal lymph node hypertrophy or splenomegaly in unirradiated hosts. Thus, IEL have the potential for proliferative activities characteristic of T cells, but they require accessory cells and/or factors such as interleukin-2 for their function in vitro and in vivo.