The liver: a model of organ-specific lymphocyte recruitment

The liver is constantly exposed to gut-derived antigens that enter via the portal vein, and it must modulate immune responses so that harmful pathogens are cleared but necessary food antigens are ignored. The liver contains a large resident and migratory population of lymphocytes and macrophages that provide immune surveillance against foreign antigen. This population of cells can be rapidly expanded in response to infection or injury by recruiting leukocytes from the circulation, a process that is dependent on the ability of lymphocytes to recognise, bind to and migrate across the endothelial cells that line the vasculature. Lymphocytes can enter the liver at several sites: the vascular endothelium in the portal tracts (comprising the hepatic artery, portal vein and bile ductule), the sinusoids (through which the blood percolates past the hepatocytes) or the central hepatic veins (through which the blood exits). The requirements and physical conditions at each site vary and there is evidence that different combinations of adhesion proteins are involved at these different sites. This article discusses the expression and function of adhesion molecules within the liver and demonstrates how specific populations of effector lymphocytes can be selectively recruited to the liver.     

The presence of specific antigen-reactive cells during the induction, recovery, and resistance phases of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is an induced disorder in which an autoimmune response specific for myelin basic protein (BP) results in neural tissue destruction and acute paralysis. Lewis rats rapidly recover from induced paralysis and do not display any clinical manifestations of EAE following a second BP injection. The object of this study was to determine if immunologic recognition of encephalitogenic fragments of the BP molecule occurs during the induction, recovery, and resistance phases of EAE. Paralysis was induced in Lewis rats by a single injection of BP in complete Freund's adjuvant (CFA). The results indicate that macrophage migration inhibition in the presence of encephalitogenic BP fragments containing amino acid residues 43–88 and 68–88 is detectable during the paralytic stage of EAE, and also following recovery from clinical neurologic impairment. Although rats which had recovered were resistant to secondary BP-induced paralysis, macrophage migration inhibition in the presence of BP, or its encephalitogenic fragments, was detected after the second BP-CFA challenge. In order to assess humoral immunologic recognition, levels of serum antibody specific for the 43–88 BP fragment were determined. Specific antibody was not detected during the paralytic episode, but appeared upon recovery. Specific antibody was also detected following the secondary BP-CFA challenge. These data indicate that antigen-sensitive cells are present during the induction, recovery, and resistance phases of EAE in the Lewis rat. The mechanism which controls the activity of these cells in vivo has not been established.     

Thymic antigen-reactive cells do not specify serological properties of antibody

Irradiated mice of the (C3H × C57BL/10)F₁ and (C57BL/6 × DBA/2)F₁ strains were reconstituted with an excess of syngeneic bone marrow cells containing precursors of immunocytes, and with graded limiting numbers of thymocytes containing antigen-reactive cells (ARC), and then injected with sheep erythrocytes. The number of ARC and their possible specialization for serological properties of antibody were investigated by determining the titer of 2-mercaptoethanol-sensitive serum hemagglutinins and hemolysins 11 days after grafting. The limiting dilution assays indicated that the number of detectable ARC/10⁶ thymocytes was of the same order of magnitude for both antibody responses. Agglutinins and lysins were associated in most recipient mice receiving an average of 1 ARC. Hence, serological properties of antibodies were not dictated by ARC, but by other cells participating in the immune responses, presumably of nonthymic origin.     

Role of interferon in lymphocyte recruitment into the skin

Large numbers of lymphocytes are recruited from the blood into sites of cutaneous DTH reactions. Our goal was to investigate the factors controlling this recruitment. 111In-labeled peritoneal exudate lymphocytes were injected iv and the accumulation of these cells in skin sites injected with a variety of stimuli, was used to measure lymphocyte recruitment in rats. Large numbers of lymphocytes migrated into vaccinia- and KLH-injected sites in sensitized animals, but only into the viral and not the KLH lesions in non-immune animals. Lymphocytes also migrated efficiently into sites injected with the alpha-interferon (IFN) inducers, uv-inactivated vaccinia virus and poly I:C, as well as into sites injected with IFN. In each case there was a dose-response relationship. Analysis of the kinetics of lymphocyte recruitment demonstrated that the peak rate of migration occurred most rapidly after the injection of IFN, later after poly I:C, and was slowest to be reached after vaccinia virus. Rabbit anti-IFN blocked the recruitment of lymphocytes by uv-inactivated vaccinia and by IFN. Histologically, all of these sites demonstrated a dense mononuclear cell infiltrate in the dermis. It is suggested that IFN may be an important mediator in the recruitment of lymphocytes into inflammatory reactions.     

Differential recruitment of PKC isoforms in HeLa cells during redox stress

The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation.     

Cell recruitment during glial repair: the role of exogenous cells

Summary Selective disruption of the neuroglia in penultimate abdominal connectives of the cockroach nerve is followed by a rapid accumulation of cells in the perineurial layer of the lesion. Subsequently, there is an abrupt, secondary, rise in cell numbers in the undamaged perineurial tissues, anterior to the lesion and adjacent to the 4th abdominal ganglia. By 7 days the increased cell numbers are again effectively confined to the original lesion zone. The initial rise in cell numbers is postulated to result from an invasion by blood-borne haemocytes and the subsequent increase, in undamaged perineurial tissues, from the mobilization of endogenous reactive cells. Recruitment of the endogenous cells is inhibited if the haemocytes are excluded from the lesion. There is a slower mobilization of sub-perineurial cells, which, again, is inhibited following exclusion of haemocytes from the lesion zone. It is postulated that the recruitment of the endogenous reactive cells is initiated by the invading haemocytes which transform to granule-containing cells and release diffusible morphogenic and/or mitogenic factors.     

The kinetics of Theileria parva infection and lymphocyte transformation in vitro

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.     

C3 receptors on human lymphocyte subsets and recruitment of ADCC effector cells by C3 fragments

The presence of C3 receptors on human peripheral blood lymphocytes (PBL) and on the ADCC-exhibiting subset (K cells) thereof was analyzed by rosetting with bovine erythrocytes (Eb) or chicken erythrocytes (Ec) carrying human C3b, C3bi, or C3d. The indicator cells were coated with 20,000 to 100,000 C3 fragments, obtained by C3 activation with purified proteins of the alternative pathway and trypsin treatment. ADCC was studied at the cellular level by means of a plaque assay, with complement-free or complement-carrying indicator cells as targets. Of the total lymphocytes, 12 to 14% bound EC3b; 6 to 8%, EC3bi; and approximately 2%, EC3d. Surface marker analysis indicated that approximately 75% of the C3b-binding lymphocytes in PBL were either B or null cells and approximately 60% of the C3bi-binding cells were T cells, as characterized by the monoclonal antibodies OKT3 and OKT4 or by presence of receptors for Helix pomatia hemagglutinin. Of the K cells, which constituted from 5 to 10% of the total lymphocytes, approximately 20% bound C3b; 30 to 35%, C3bi; and 7 to 8%, C3d. Here the majority of the C3b binders were null cells, and the majority of the C3bi and C3d binders were T cells. Only one-third of the C3b-binding K cells and one-fifth of the C3bi-binding K cells bound both fragments. The nature of these double binding cells is unknown. In contrast, all C3d-binding K cells bound C3bi as well. C3 fragment-carrying target cells did not induce K cell-mediated lysis in the absence of anti-target antibodies but strongly enhanced ADCC in the presence of sublytic concentrations of such antibodies. The rank order for C3 fragment-induced enhancement was C3bi greater than C3d greater than C3b. It reflected the relative proportions of effector cells binding the different fragments. Enhancement was the expression of effector cell recruitment rather than of increased cytolytic activity of individual K cells. This recruitment was selective in that C3b-carrying target cells primarily recruited effector cells of null type, binding C3b, while C3bi- or C3d-carrying targets primarily recruited C3bi and/or C3d-binding K cells of T gamma type. Thus, these experiments show directly at the effector cell level that cell-bound C3 fragments constitute important recognition structures, which strongly amplify ADCC both by recruiting the proper effector cells into the cytolytic reaction and by very significantly decreasing the antibody concentration needed for its induction.     

The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.     

The use of immunological tolerance to investigate B lymphocyte replacement kinetics in chickens

A simple mathematical model has been derived, describing the irreversible inactivation of immature B cells by high doses of antigen during induction of tolerance, and the antigen-independent replacement of B cells by differentiation of their precursors. The latter leads to recovery from tolerance, the rate of which can be used to assess the rate of B cell replacement in experiments. The model has been compared with experimental tolerance to human albumin in newly hatched chickens.(1) It has been shown that this tolerance cannot be explained only by elimination of B cells but (2) the computed rate of B cell replacement agreed with the experimental rate assessed by immunization of tolerant chickens with a cross-reacting antigen. (3) In order to further verify the model, additional experiments to test the rate of B cell replacement were suggested by the model.     

The effects of major and minor trauma on lymphocyte kinetics in mice.

The effects of major and minor trauma on the circulating white blood cell populations of C57BL mice were followed. The results showed that not only major trauma (nephrectomy) but minor injury and stress (e.g. injection, bleeding) triggered a highly significant fall (50-70%) in the number of lymphocytes circulating in the blood. The fall was a gradual one, with the maximal drop 2 h after the operation or handling procedure. Major trauma resulted in a fall in both B and T lymphocytes. Minor trauma produced a fall in B lymphocytes only. A 3-4 fold increase in circulating polymorph numbers also accompanied major trauma, but no increase was observed after minor trauma. The blood picture returned to normal generally within 24 h of both minor and major trauma. Repetition of the trauma stimulus after recovery led to a renewed trauma response. Bilateral adrenalectomy abolished the lymphocyte response to major and minor trauma and decreased the polymorph response to major trauma by more than 50%, indicating that stress hormones played a role in these changes. Studies with 51chromium-labelled lymphocytes, transferred into traumatized and adrenalectomized animals, suggested that decreased entry of lymphocytes into the blood (rather than increased exit from the blood into the tissues, or cell death) was the most likely mechanism of the lymphopenia following trauma.     

Imprinting the fate of antigen-reactive B cells through the affinity of the B cell receptor

Long-lived plasma cells (PCs) and memory B cells (B(mem)) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short- or long-lived PCs and B(mem). Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither B(mem) nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but B(mem) remain extinct. In contrast, lower affinity interactions show tempered GCs, producing B(mem) and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity.     

Secretory kinetics in the follicular cells of silkmoths during eggshell formation

Procedures for quantitative autoradiography were used for studying the process of secretion of eggshell (chorion) proteins in the follicular epithelium of silkmoths. The method was based on photometric measurements of the reflectance of vertically illuminated autoradiographic silver grains. Results were analyzed and plotted by computer. Secretory kinetics were also determined by analysis of labeled proteins in physically separated epithelium and chorion. Rapid accumulation of radioactivity into "clumps" visualized by light microscope autoradiography and evidence from preliminary electron microscope autoradiography indicate that, within 2 min from the time of synthesis, labeled chorion proteins move to Golgi regions scattered throughout the cytoplasm. The proteins begin to accumulate in the apical area 10-20 min later and to be discharged from the cell. The time for half-secretion is 20-25 min, and discharge is essentially complete 30-50 min after labeling. At the developmental stages examined, the kinetics of secretion appear to be similar for all proteins. Within the chorion the proteins rapidly assume a characteristic distribution, which varies for different developmental stages. Two relatively slow steps have been identified in secretion, associated with residence in Golgi regions and in the cell apex, respectively. By contrast, translocation of proteins across the cell and deposition of discharged proteins in the chorion are rapid steps.     

Identification of circulating human antigen-reactive CD4+ FOXP3+ natural regulatory T cells

Circulating human CD4(+)CD25(++)CD127(-)FOXP3(+) T cells with a persistent demethylated regulatory T cell (Treg)-specific demethylated region Foxp3 gene are considered natural Tregs (nTregs). We have shown that it is possible to identify functional Ag-reactive nTregs cells for a range of different common viral and vaccination Ags. The frequency of these Ag-reactive nTregs within the nTreg population is strikingly similar to the frequency of Ag-reactive T effector cells within the CD4(+) T cell population. The Ag-reactive nTregs could be recognized with great specificity by induction of CD154 expression. These CD154(+) Ag-reactive nTregs showed a memory phenotype and shared all phenotypical and functional characteristics of nTregs. The isolated CD154(+) nTregs could be most efficiently expanded by specific antigenic stimulation, while their Ag-reactive suppressive activity was maintained. After an in vivo booster Ag challenge, the ratio of Ag-reactive T cells to Ag-reactive Tregs increased substantially, which could be attributed to the rise in effector T cells but not Tregs. In conclusion, the nTreg population mirrors the effector T cell population in the frequency of Ag-reactive T cells. Isolation and expansion of functional Ag-reactive nTregs is possible and of potential benefit for specific therapeutic goals.     

Involvement of intestinal epithelial cells in dendritic cell recruitment during C. parvum infection

Dendritic cells (DCs) play a key role in activating and orientating immune responses. Little is currently known about DC recruitment during Cryptosporidium parvum infection. In the intestine, epithelial cells act as sensors, providing the first signals in response to infection by enteric pathogens. We analyzed the contribution of these cells to the recruitment of DCs during cryptosporidiosis. We found that intestinal epithelial cells produced a broad range of DC-attracting chemokines in vitro in response to C. parvum infection. The supernatant of the infected cells induced the migration of both bone marrow-derived DCs (BMDC) and the SRDC lymphoid dendritic cell line. Chemokine neutralization abolished DC migration in these assays. We next analyzed chemokine mRNA expression in the mucosa of C. parvum-infected neonatal mice and recruitment of the various subsets of DCs. Myeloid (CD11c+ CD11b+) and double-negative DCs (CD11c+ CD11b- CD8alpha-) were the main subsets recruited in the ileum during C. parvum infection, via a mechanism involving IFNgamma. DCs were also recruited and activated in the draining lymph nodes during C. parvum infection, as shown by the upregulation of expression of MHC II and of the costimulation molecules CD40 and CD86.