Induction and recall in contact sensivitity. Changes in skin and draining lymph nodes of intact and thymectomized mice.

The cellular events in the ear skin and draining lymph node during the induction of contact sensitivity to 2-ethoxy methylene-5-oxazolone (oxazolone) have been studied in three strains of mice. The principal findings in the skin during the first 24 hr were invasion of polymorphs and destruction of pilosebaceous units, in both intact and thymectomized mice. Subsequently, the dermal cellular infiltrate increased and there was acanthosis of the epidermis. No lymphocytes were seen in the dermis or penetrating the epidermal basal cell layer in thymectomized mice. During the first 24 hr in the draining node, polymorphs and macrophages bearing a pigment with staining properties similar to melanin were seen in the marginal and medullary sinuses, in intact and thymectomized mice. Major differences, however, were revealed during 2-4 days when massive proliferation of large pyroninophilic blast cells occurred in the thymus-dependent area of the nodes from intact mice only. On testing, there was a prompt, measurable increase in ear thickness only in intact mice. This increase reached a peak at 24 hr - typical of a delayed type reaction. At testing, the ears from intact mice showed epidermal vesiculation and a considerable dermal cellular infiltrate with a substantial number of lymphocytes. This was in contrast with the completely quiescent appearance of the ear skin of thymectomized mice. Finally, we have discussed the use of the mouse as an experimental tool for studying contact sensitivity and have analyzed the role of the thymus-derived lymphocyte and the site where it becomes sensitized, in the light of current theory on the origin of cells and site where sensitization takes place in cell-mediated reactions.

Regional blood flow and its relationship to lymphocyte and lymphoblast traffic during a primary immune reaction.

The relationship of alterations in blood flow with changes in cell distribution has been studied in an inflammatory site and its draining lymph node during the induction of an immune reaction with oxazolone in mice. The cells which move to the site of inflammation are predominantly lymphoblasts and their increased localization in the inflamed ear is significantly correlated with increased regional blood flow to the inflamed tissue. The existence of this correlation is not antigen dependent although there is a relative increment of lymphoblasts which are specifically primed to the inflammatory agent. The localization of nonblastic (small) 51Cr-labeled lymphocytes on the other hand is substantial only in lymphoid tissue and during the induction of an immune reaction after oxazolone application, the increase in localization of these cells in the draining lymph node is positively correlated with increased blood flow to the node. Furthermore, the probability of finding 51Cr labeled lymphocytes in a particular lymph node is related to the regional blood flow which that node receives.

Antigen-induced locomotor responses in lymphocytes.

The effect of protein antigens on the locomotion of lymphocytes from the lymph nodes draining the site of antigenic challenge in immunized mice, and from the same nodes in control mice, was studied in filters using a checkerboard assay in which the absolute concentration and the concentration gradient of attractant was varied in a series of chambers. Serum albumin (HSA or BSA) was chemokinetic for unimmunized lymphocytes inasmuch as the distance migrated into filters by cells in its presence varied with the absolute concentration of albumin, but not with the concentration gradient, indicating an influence of the serum albumin on the rate but not on the direction of locomotion. Ovalbumin and nonalbumin proteins did not show this effect. Using the same assay, the migration of primed lymphocytes in the presence of the priming antigen was shown to be influenced by the antigen gradient in a way that suggested a positive chemotactic response of the lymphocytes to antigen. This response was only shown clearly when the cells were in a chemokinetic medium containing serum albumin.

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.

Hypersensitivity reactions in the small intestine. VII. Induction of the intestinal phase of murine graft-versus-host-reaction by Lyt 2- T cells activated by I-A alloantigens.

This study has examined the nature of the T lymphocytes and the alloantigens that induce the intestinal phase of graft-versus-host reaction in unirradiated F1 mice. Parental spleen cells were depleted of T cells subsets by treatment with anti-Lyt monoclonal antibodies and complement, and we show that Lyt 2- cells alone induce the increased lymphocytic infiltration of the epithelium that characterizes the intestinal graft-versus-host reaction. Lyt 2- cells are also required to induce some of the associated crypt hyperplasia, but the full crypt changes require both Lyt 2- and Lyt 2+ T cells. In intra-H-2 recombinant congenic F1 mice with graft-versus-host reaction, a disparity at the I-A locus was alone sufficient and necessary for crypt hyperplasia and increased intraepithelial lymphocyte counts, while an I-J incompatibility led to suppression of both these indices. The results support the hypothesis that the intestinal pathology of acute GVHD is induced by class II MHC-restricted delayed-type hypersensitivity effector T cells.

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.

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.

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.

Intestinal mast cell response in thymectomised and normal mice infected with Trichinella spiralis.

In NIH mice, expulsion of Trichinella spiralis from the small intestine and increase in intestinal mast cells were each dependent on the presence of T-lymphocytes. Both changes were deficient in thymectomised mice but could be largely restored by reconstitution of thymectomised mice with syngenetic mesenteric lymph node cells. In both NIH and BALB/C mice the majority of the increased number of mast cells occurred within the intestinal epithelium. In NIH mice increase in the number of intestinal mast cells coincided roughly with expulsion of the parasites. In BALB/C mice increase in the numbers of intestinal mast cells did not appear to be connected with the location or expulsion of parasites and it is concluded that mast cell proliferation, accumulation and discharge per se do not result in or from worm expulsion.

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.

Regional blood flow and the localization of lymphoblasts in the small intestine of the mouse. I. Examination of normal small intestine.

The localization of 125I-UdR-labelled mesenteric lymphoblasts and the fraction of the cardiac output delivered to the small intestine was investigated in mice. When different regions of the small intestine were examined, the proportional delivery of the cardiac output and the localization of lymphoblasts were found to vary along the length of the small intestine. A significant correlation between these two phenomena was identified when both lymphoblast localization and the distribution of the cardiac output within the small intestine were studied concurrently. The intestinal localization of populations of unseparated or T-enriched mesenteric lymphoblasts and peripheral lymphoblasts all showed a similar degree of correlation with the fraction of the cardiac output delivered along the small intestine in spite of marked differences in their proclivity to accumulate in the gut. We conclude that there is an important relationship in normal animals between the level of lymphoblast accumulation within a particular region of the small intestine and the delivery of blood-borne cells to that region. This relationship could provide a physiological explanation for a antigen-independent yet non-uniform distribution of effector cells within the lamina propria of unimmunized animals.

Regional blood flow and the localisation of lymphoblasts in the small intestine of the mouse: effect of an elemental diet.

To test the hypothesis that food antigens influence the in vivo migration of lymphoblasts to the small intestine, the effect of an elemental diet (Vivonex) on the distribution of lymphoblasts within the small intestine of mice has been examined. Viable lymphoblasts from the mesenteric nodes of conventionally fed animals were labelled in vitro and given intravenously to recipient mice fed either a standard diet or elemental diet. The localisation of these cells within the small intestine was altered in the animals fed the elemental diet but only in the distal half of the small intestine. The relationship of the localisation of blast cells to the delivery of cardiac output along the small intestine was examined by assessing cell localisation in conjunction with the distribution of an isotopic indicator (86RbC1). The results show that the pattern of localisation of lymphoblasts within the small intestine is related to the probability that they will be delivered to different regions by the blood stream. Therefore, the alterations in blast localisation in the small intestine of animals of the elemental diet can be viewed as a consequence of changes in the perfusion of the distal small intestine. These results do not support the concept that antigens directly influence the efficiency with which blast cells migrate into the intestinal mucosa.

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.