Migration of thymus cells to the developing gut-associated lymphoid tissues of the young rabbit

Thymus cell migration to the gut-associated lymphoid tissues (GALT) as compared to other lymphoid tissues in young rabbits was determined following in vivo intrathymic inoculation of tritiated thymidine. The GALT received as many or more thymus cells than the spleen or lymph nodes during the first few postnatal days. Migration to the GALT and nonGALT decreased with age, and seeding appeared to be essentially complete by 30–40 days.     

CD5+ B cells predominate in peripheral tissues of rabbit.

Restricted usage of VH genes is observed in rabbit B lymphocytes and in human and murine CD5 B lymphocytes. This observation raised the possibility that most rabbit B lymphocytes were CD5+. To investigate this we cloned the CD5 gene from a rabbit cosmid library, using a probe derived from human CD5 cDNA. The rabbit CD5 gene was transfected into a murine T cell line and then we used the transfectants to develop anti-rabbit CD5 mAb. By Western blot analysis, the mAb reacted with a 67-kDa protein in lysates prepared from mesenteric lymph node and spleen cells. We determined the frequency of CD5+ B lymphocytes in peripheral lymphoid tissues of adult rabbits by two-color immunofluorescence analysis using anti-CD5 mAb and anti-L chain antibodies. The analysis showed that essentially all peripheral B lymphocytes in adult rabbits express CD5. The observation that CD5 is expressed on nearly all rabbit B lymphocytes contrasts markedly to mouse and human, where only a small number of B lymphocytes express CD5. We propose that most peripheral B lymphocytes in rabbit, as in chicken, develop early in ontogeny and are maintained throughout life by a self-renewing process.     

Generation and characterization of monoclonal antibodies recognizing follicle epithelial M cells in rabbit gut-associated lymphoid tissues

A panel of mouse B cell hybridomas producing monoclonal antibodies (mAb) directed against rabbit M cell-containing epithelia was developed. By immunohistochemistry, the mAb 5D9, 5B11, 1D9, and 4G2 were found to label approximately 50% of the follicle-associated epithelial (FAE) cell populations overlying lymphoid follicles in Peyer's patches, cecal patch, sacculus rotundus, and appendix. The cell staining was localized to FAE cell basolateral surfaces outlining the M cell pockets which enclosed clusters of mononuclear leukocytes, and extended from the crypts of Peyer's patches and sacculus rotundus, and appendiceal crevices, to the apices of domes. In contrast, the stem cell and proliferative regions facing the lamina propria were devoid of immunologically reactive sites. The mAb 5D9, 1D9, and 4G2 did not recognize antigens associated with non-FAE cells in the intestinal lymphoid tissues examined. Only the mAb 5B11 labeled apical surfaces of Peyer's patch and cecal patch non-FAE. However, this mAb did not label interdomal colonic epithelial cells in sacculus rotundus and appendix. Besides recognizing FAE cells, the mAb 4G2 recognized a cross-reactive antigen displayed by dome and lymphoid follicle lymphocytes. By flow cytometry, the mAb 5D9, 5B11, and 1D9 were shown to stain from 14 to 29% of the cells in M cell-enriched populations prepared from Peyer's patches, sacculus rotundus, and appendix, whereas mAb 4G2 was found to recognize 44-54% of the cells. Two-color flow cytometric analysis showed that the mAb stained a functionally distinct subpopulation of Peyer's patch phagocytic cells and did not recognize spleen macrophages. These findings indicate that the panel of mAb recognized novel antigens expressed by FAE cells overlying intestinal lymphoid aggregates, and that the mAb allow identification of phagocytic M cells in suspensions of FAE cells.     

Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs

Background

Buruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear.

Methodology/Principal Findings

Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes.

Conclusion

In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.     

Migratory patterns of B lymphocytes. I. Fate of cells from central and peripheral lymphoid organs in the rabbit and its selective alteration by anti-immunoglobulin.

Homing properties of ³H-adenosine-labeled bone marrow (BM), appendix (App), and mesenteric (Mes) and popliteal (Popl) lymph node (LN) cells were studied in the rabbit. While BM cells 24–48 hr after transfer produced equivalent radioactivity (cpm/mg) in App, Mes, and Popl LN of recipients, App and Popl LN cells did not, showing a highly significant preference for their organ of origin. “Homing to antigen” was shown if Popl LN cells were taken from rabbits immunized to the same antigen as was injected in one footpad of recipients 2 weeks earlier.Pretreatment of App cells with sheep anti-rabbit Ig abolished their preferential localization in App as judged in recipients killed 5 hr after transfer, but this was a transient effect and no longer demonstrable by 24 hr.Histological localization of labeled cells showed the corona of follicles in the center of dome-shaped areas of the App to represent “B-influx areas” after transfer of all cell types and this localization was blocked temporarily (5 hr) by prior incubation of App cells with anti-Ig. Thymus-dependent interfollicular areas showed labeled cells after LN cell transfer, and less after App cells, but none after BM. Emphasized in the discussion are (i) the possible effect of antigen on homing of B cells and (ii) the implications of the findings with respect to the appendix as a peripheral lymphoid organ.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a "lacy" pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macrophages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Interdigitating cells in the lymphoid tissues of bovine fetuses and calves

Summary Electron-microscopic studies of lymphoid tissues from bovine fetuses and from calves disclosed a non-lymphoid cell type in the thymus-dependent zones of secondary lymphoid tissues and in the thymus that is distinguishable from reticulum cells, epithelial and endothelial cells, and macrophages. Based on morphological and topographical criteria, the cell is identified as the interdigitating cell. In addition, studies of the tissues of normal and virus-challenged fetuses, and of conventionally reared calves, indicated that the interdigitating cells originate from monocytoid cells, which undergo differentiation in the thymus-dependent zones during an immune response.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Summary Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a lacy pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macropages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Cutaneous lymphocyte antigen is expressed on memory/effector B cells in the peripheral blood and monocytoid B cells in the lymphoid tissues.

Cutaneous lymphocyte antigen (CLA) is expressed on a subpopulation of human memory T cells and is involved in the primary step of their skin homing. T cells and some B cells in the peripheral blood express CLA, but the pathophysiologic roles of CLA(+) B cells have not yet been clarified. We examined the relationships among CLA expression in B cells and immunoglobulin heavy chain subtype, the localization of CLA(+) B cells in the peripheral lymphoid tissues, and their functional binding to E-selectin. CLA was expressed on class-switched, memory B cells in the peripheral blood and tonsils as revealed by flow cytometry. Immunohistochemical staining of the lymph nodes with various types of inflammation or reactive hyperplasia showed CLA on the monocytoid B cells, which correspond to memory cells. The functional study revealed that CLA on B cells bound to E-selectin transfectants. E-selectin was detected on some of the high endothelial venules in the monocytoid B-cell-rich lymph nodes. These findings suggest that CLA is also expressed on a subset of memory/effector B cells, in addition to a subset of memory T cells. Such B cells were located in the lymph nodes or tonsils and rarely in chronic dermatitis. Therefore, CLA seems to be related to memory/effector B-cell trafficking to the lymph nodes or tonsils. According to the multistep theory, mechanisms involved in the second or third step might be different between CLA(+) B and T cells.     

Rosette formation of guinea pig lymphoid cells with rabbit erythrocytes—Differences between thymocytes and peripheral blood lymphocytes

Rosette formation of guinea pig thymocytes (Th) and thymus-derived peripheral blood lymphocytes (TBL) was tested under different experimental conditions. Up to 93% of Th and 38% of TBL showed an affinity to rabbit red blood cells (RRBC). Treatment with metabolic inhibitors like sodium azide and sodium cyanide or freezing and thawing nearly abolished rosette formation by TBL but was ineffective with respect to Th. Heating the cells destroyed rosette-forming capacity of both cell types. These results indicate that spontaneous rosette formation with RRBC by Th does not require the live cell.     

Localization of PNA-binding and nonbinding thymus cells in peripheral lymphoid organs

Thymus cells were labeled in vitro with FITC and injected into syngeneic recipients. In cell suspensions of lymphoid organs green cells were inspected for PNA receptors with double immunofluorescence. A striking preference of PNA-negative cells to localize in lymph nodes and the lymphoid compartment of the spleen was demonstrated. Incubation with anti-Ly sera revealed that Ly 1⁺ PNA-negative cells homed in popliteal lymph nodes and Peyer's patch but not in mesenteric lymph nodes.     

Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the Syrian hamster

In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks.     

Purine nucleoside kinases in mouse tissues and human lymphoid cells in culture

• 1.1. Purine nucleoside kinase activities (adenosine kinase, deoxyadenosine kinase and arabinosyl adenine kinase) in mouse tissues were in the following order: liver > kidney > heart > lung > brain > spleen > intestine.
• 2.2. Ratios of deoxyadenosine or arabinosyl adenine kinase to adenosine kinase were significantly higher (10–200 fold) in human lymphoid cells than in mouse tissues.
• 3.3. Leukocytes from T-cell acute lymphoblastic leukemic patients had 5–20 fold elevated adenosine deaminase as compared to normal T-cells.