Characterization of monolayer and organ cultures of cloned and enriched lymphohematopoietic stromal cell populations

The role of hematopoietic microenvironments in the regulation of maturation and differentiation of hematopoietic cells, although heavily debated, remains uncertain. Several investigators have suggested that the adherent “stromal” cell populations, which grow as colonies in cultures of lymphomyeloid tissues, include the cells involved in such regulatory processes. Grossly, the colonies described by several investigators appear similar morphologically, and the cells giving rise to them have been variously termed (1) fibroblast colony forming cells (FCFC), (2) plaque forming units-culture (PFU-C), (3) macrophage colonies, and (4) marrow stromal cells. FCFC have been reported to re-establish their parent microenvironment when transplanted in an allogeneic system. In this study, cloned and enriched cell populations obtained from such colonies in cultures of murine lymphomyeloid tissues have been characterized by their growth in culture and using morphological, histochemical, and electron microscopic techniques. The results demonstrated that, although the initial stromal colonies appeared to be identical, the constituent cell types varied considerably. Some colonies were comprised primarily of macrophages, while others appeared to contain predominantly fibroblasts; two additional cell types that established colonies have not yet been satisfactorily identified. These results demonstrate the heterogeneity of lymphomyeloid stromal colonies. There is a need for caution in the analysis of experiments in which uncharacterized stromal cell colonies are transplanted or employed as supporting monolayers in culture systems in experiments designed to evaluate the origins and functions of lymphohematopoietic stroma.     

Morphological relationships and leukocyte influence on steroid production in the epigonal organ-ovary complex of the skate, Leucoraja erinacea

In elasmobranchs, a unique association exists between an immune tissue, the epigonal organ (EO), and the gonads. In this study, the histological and vascular relationships of the EO and ovarian follicles of the little skate, Leucoraja erinacea, were assessed. Perfusions of Evans blue dye and Batson's monomer showed a shared vascular pathway from the gonadal artery into the epigonal-ovary complex, with blood first entering the EO and then perfusing the ovarian follicles. Histological studies demonstrated direct cellular contact between epigonal leukocytes and the follicle wall (FW), as well as the presence of leukocytes between the steroidogenic theca and granulosa cells. In vitro analyses demonstrated that epigonal cells co-cultured with FW cells cause a dose-dependent inhibition of estrogen (E2) and testosterone (T) production. In contrast, conditioned media from epigonal leukocytes, stimulated or unstimulated with lipopolysaccharide (10 microg/ml), increase the production of E2 and T from FW cells of the ovaries. These studies provide a basis for further investigations of leukocyte secreted factors and cell contact modulation of follicular steroid production.     

Marker Chromosome Analysis of Tetraparental AKR↔CBA-T6 Mouse Chimaeras

Marker chromosome analysis of 18 tetraparental AKR↔CBA/H-T6 chimaeras revealed a great excess of AKR mitoses over CBA mitoses in direct preparations from lymphomyeloid tissues, corneal epithelium, intestinal epithelium and skin (Table 1). The degree of AKR dominance was strongly influenced by anatomical site (Tables 2 and 3). The testes of three out of four XY/XY males contained a marked excess of AKR germ cells and produced a parallel excess of functional AKR gametes (Table 4). Mitotic spreads in mitogen-stimulated cultures of tail blood were overwhelmingly of AKR type in 1972, but less so in 1973 (Table 5).
The coat phenotypes, and breeding results from known or presumptive XX/XX females, suggest that AKR and CBA cells were numerically balanced when melanoblasts and oocytes were formed during embryonic development, and therefore that the striking deviations from equality observed in mitotic populations of adult chimaeras arose later by differential proliferation or survival of AKR cells, or both.
The low frequency of lymphomas, compared with normal AKR mice, previously reported in these chimaeras cannot therefore be accounted for by insufficiency of AKR cells in the thymus or elsewhere in the lymphomyeloid tissues. One of three lymphomas studied was CBA type. This suggests that the high risk of lymphomatous transformation is not an autonomous property of AKR cells.     

Development of granulocytes in haematopoietic tissues of Rhizoprionodon lalandii

Granulocytes of the epigonal and Leydig organs of Rhizoprionodon lalandii were identified and classified into three different cell types, type I and type II eosinophils and neutrophils. The development of these cells in the haematopoietic tissues was dynamic, demonstrated by nuclear immunopositivity for the proliferating cell nuclear antigen (PCNA) proteins and was regulated by various cytokines, including the transforming growth factor β -l (TGF/ β ₁). The expression pattern of these cells was heterogeneous among individual cells and TGF/ β ₁-immunostaining was found principally in the cytoplasm of immature granulocytes. The presence of TGF/ β ₁ in cells about to divide was demonstrated suggesting that modulation of differentiation and proliferation occurs in the haematopoietic tissues of this species of elasmobranch.     

An ultrastructural and immunocytochemical study on the lymphomyeloid tissue in the embryonic kidney of the dogfish, Scyliorhinus canicula

The central role of the kidney during early development in the dogfish, Scyliorhinus canicula , is haemopoietic. The kidney is the first tissue to become lymphomyeloid and lymphocytes and developing granulocytes are found in the interstitial tissue surrounding the renal tubules. The lymphomyeloid role of the kidney is transient and does not persist after hatching. The kidney is a major immunoglobulin (Ig) containing tissue during early development as evidenced by increasing numbers of Ig–positive cells.     

Hibernation alters the frog's immune system.

The lymphomyeloid organs and blood leukocyte populations of the leopard frog, Rana pipiens, undergo conspicuous changes during hibernation at 4 degrees C. Within the blood, spleen, thymus, jugular bodies, and bone marrow there was a progressive loss of hemopoietic populations resulting in a marked lymphocyte depletion. Termination of the 135-day hibernation period resulted in the restoration of all hemopoietic elements in the blood and lymphomyeloid organs within 30 days. Frogs subjected to experimental hibernation and immunized showed weakened immune responses when brought from the hibernaculum. Plaque-forming cells (PFC) were lower in spleen, jugular bodies, and bone marrow, and serum antibody titers were also lower. Although the kinetics of the primary responses were essentially the same, the secondary responses differed suggesting major rearrangements with respect to the numbers of cells and their function in secreting antibody. The apparent lymphocyte aplasia may contribute to the absence of immunological responsiveness during periods of hibernation.     

Composition and ultrastructure of elasmobranch granulocytes. III. Sharks (Lamniformes)

The peripheral blood granulocyte composition of five species of shark is given together with ultrastructural observations made on the epigonal organ and blood of Mustelus lenticulatus and spleen of Apristurus sp. Three granulocyte lineages occurred in Mustelus . Ultrastructurally, eosinophilic granulocytes contained granules that were very variable in size and contained parallel fibrillar arrays that were unidirectional in small granules, but which were often orientated in several directions in larger granules. This dense core material sometimes formed angular crystalloids.
Eosinophils had ovoid or irregular granules that were usually uniformly electron-dense, but some had an eccentric ovoid area of greater electron-density. Some eosinophils enter the blood where they are phagocytic, but others, in blood and epigonal organ, showed an unusual but characteristic process of disintegration, leaving a ragged cell full of cellular debris.
The third granulocyte type had features of mast cells and basophils of higher vertebrates and was tentatively identified as belonging to that lineage. The inter-relationships of these cells, and of them with granulocytes of other elasmobranchs, are discussed.     

Members of the Ikaros gene family are present in early representative vertebrates

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.     

Size Relations of Lymphomyeloid Organs in Some Cartilaginous Fish

In elasmobranchs (sharks, rays) the total weight of lymphomyeloid structures isolated by dissection varied between about 0.6 and 1.0% of the body weight. In a holocephalan (Chimaera) the corresponding value was 1.7%. Reciprocal size relations may exist between different types of lymphomyeloid organs. A thymus was found in adult specimens of rays (Raja) and the rabbit-fish (Chimaera). The lymphomyeloid organs supposedly are active in haemopoiesis and the immune responses.     

Decidual cell-specific surface antigen(s) recognized by monoclonal antibodies: tissue and species distribution

Decidual cells are direct descendants of endometrial stromal cells and the ultimate progeny of bone marrow-derived precursors. In view of their bone marrow genealogy and demonstrated immunoregulatory role during pregnancy, this study attempted to identify a lineage-specific differentiation marker(s) on murine decidual cells with the hope of tracing their developmental pathway and exploring their familial relationship to other lymphomyeloid cells. Two protein A-binding, IgG2b isotype monoclonal antibodies (secreted by clones 16F12 and 2G4F8) were raised by immunizing virgin CBA mice with syngeneic decidual cells. The presence and the density of the antigenic marker(s) recognized by these antibodies were examined by radioautography on various cell types in single cell suspensions of the decidua, placenta, and lymphomyeloid organs after a sandwich labeling with hybridoma supernatants followed by 125I-protein A. Both antibodies appeared to recognize antigen(s) unique for the decidual cell lineage in mice, humans, and rats. The incidence of antigen-bearing decidual cells increased with gestational age in CBA, C3H, and CD1 mice between days 8 and 14, and in humans between 6 and 10.5 wk; in rats, however, some decline was noted between days 8 and 14. The binding was always higher with 16F12 than with 2G4F8 supernatants. No significant binding of either antibody to trophoblast cells of the placenta or leukocytes within the decidua was noted in any of the above mouse strains or species. Little or no labeling of any cell type was seen on lymphomyeloid cells of the virgin or pregnant CBA mice, but a consistent labeling of a rare blast-type cell in the blood was observed with both antibodies, raising the possibility that this cell may represent the circulating precursor of the decidual cell lineage. It remains to be investigated whether these antibodies are recognizing the same or different differentiation antigen(s) on the decidual cells, and whether a conservation of this antigen(s) during speciation signifies its functional importance.     

Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health

Reports that elasmobranchs (sharks, skates, and rays) may havea low incidence of disease have stimulated interest in understandingthe role of their immune system in this apparent resistance.Although research in this area may potentially translate intoapplications for human health, a basic understanding of theelasmobranch immune system components and how they functionis essential. As in higher vertebrates, elasmobranch fishespossess thymus and spleen, but in the absence of bone marrowand lymph nodes, these fish have evolved unique lymphomyeloidtissues, namely epigonal and Leydig organs. As conditions forshort-term culture of elasmobranch immune cells have becomebetter understood, the opportunity to examine functional activityof cytokine-like factors derived from conditioned culture mediumhas resulted in the identification of growth inhibitory activityagainst a variety of tumor cell lines. Specifically, the mediumenriched by short term culture of bonnethead shark (Sphyrnatiburo) epigonal cells (epigonal conditioned medium, ECM) hasbeen shown to inhibit the growth of mammalian tumor cell lines,including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-celllymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer(PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinomacell lines (MCF7, HCC38, Hs578T). Of the cell lines tested,WEHI-164, A375.S2, Daudi, and Jurkat cells were among the mostsensitive to growth inhibitory activity of ECM whereas PANC-1and NIH:OVCAR-3 cells were among the least sensitive. In addition,ECM demonstrated preferential growth inhibition of malignantcells in assays against two different malignant/non-malignantcell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separationof protein components of ECM using SDS-PAGE resulted in a veryreproducible pattern of three major bands corresponding to molecularsizes of approximately 40–42 kD, 24 kD, and 17 kD. Activityis lost after heating at 75°C for 30 min, and can be diminishedby treatment with proteinase K and protease. Activity is notaffected by treating with trypsin, DNase I or RNase A.     

In vivo exposure of clearnose skates, Raja eglanteria, to ionising X-radiation: acute effects on the peripheral blood, spleen, and epigonal and Leydig organs

The effects of ionising radiation on the peripheral blood, spleen, and epigonal and Leydig organs of cartilaginous fishes were investigated using juvenile clearnose skates, Raja eglanteria. Skates (N = 80) were sacrificed 12 days after exposure to 0-75 Gy of X-radiation, and morphometrics (body mass, disc width, total length), mass of spleens and epigonal organs, and peripheral blood leucocyte (PBL) counts were compared to controls using ANOVA. Spleen and epigonal organ mass and PBL counts declined logarithmically as a function of radiation dose. To assess recovery from X-radiation, skates (N = 40) were exposed to 0, 9 or 18 Gy and sacrificed when moribund or on days 10, 20, 30 and 40 post-irradiation. Partial recovery of Leydig organ and splenic red pulp was evident after 40 days in skates exposed to 9 Gy, but no indication of recovery was apparent at higher doses. Median lethal dose by 30 days (LD50/30) was calculated to be 9-18 Gy, similar to that determined for other fishes.     

Histogenesis of the lymphomyeloid complex in the larval leopard frog, Rana pipiens

A histological study was undertaken of the differentiation of the lymphomyeloid complex of larvae of the common leopard frog, Rana pipiens, reared at 18–21 °C. Immature lymphocytes are first recognised in the thymus and pronephros at stage I of Taylor and Kollros ('46). By the end of stage II, small lymphocytes are regularly found among the predominant larger lymphoid cells in the thymus, in which corticomedullary differentiation has begun. At this time, a few small lymphocytes are also apparent in the ventral cavity bodies, lymph gland, pronephros, mesonephros and intestine, but rarely occur in the spleen. During stages III and IV extensive development of these components of the lymphomyeloid complex occurs. The organs now contain large numbers of mature lymphocytes and have attained states of differentiation that remain essentially similar in subsequent larval stages. By stage V, small epithelium-associated lymphoid accumulations are abundant throughout the length of the gastrointestinal tract. The anlagen of the procoracoid body and bone marrow appear just prior to metamorphosis. The kidneys are the main sites of blood formation in the larva. Masses of granulocytes are also usually found in the abundant ventral cavity bodies. A consideration of the roles of each of the organs provides insight into the ontogeny of the immune system of the Anura in general.     

Lymphoid Organs and Blood Cells of the Caecilian Ichthyophis kohtaoensis

The present work analyzes by light microscopy circulating blood cells and lymphoid organs of the caecilian, ichthyophis kohtaoensis. Peripheral blood contains erythrocytes, thrombocytes, lymphocytes, monocytes, heterophils, easinophils and basophils. Thymus, liver and spleen are the major lymphomyeloid organs in this caecilian and there is not a haemopoietic functioning bone marrow. The results are discussed on the basis of the possible phylogenetic positions of the Apoda emphasizing their relationships to the Urodela.     

Heterogeneity of the tolerant state in rats with long established skin grafts

Tolerance in rats with long established allogeneic skin grafts brought about by the neonatal inoculation of hybrid lymphomyeloid cells is immunologically heterogeneous because 2 states of tolerance may be defined operationally as fully and partially tolerant. T cells from fully tolerant donors transfer tolerance and are unresponsive in MLC, but T cells from partially tolerant donors do not transfer tolerance and are modestly responsive in MLC. A state of tolerance may exist without detectable suppression. Therefore, tolerance, traditionally defined as the permanent acceptance of an allogeneic skin graft, does not reflect a uniform immunologic state.     

Adiponectin, a fat cell product, influences the earliest lymphocyte precursors in bone marrow cultures by activation of the cyclooxygenase-prostaglandin pathway in stromal cells

Adiponectin, an adipocyte-derived hormone, is attracting considerable interest as a potential drug for diabetes and obesity. Originally cloned from human s.c. fat, the protein is also found in bone marrow fat cells and has an inhibitory effect on adipocyte differentiation. The aim of the present study is to explore possible influences on lymphohematopoiesis. Recombinant adiponectin strongly inhibited B lymphopoiesis in long-term bone marrow cultures, but only when stromal cells were present and only when cultures were initiated with the earliest category of lymphocyte precursors. Cyclooxygenase inhibitors abrogated the response of early lymphoid progenitors to adiponectin in stromal cell-containing cultures. Furthermore, PGE(2), a major product of cyclooxygenase-2 activity, had a direct inhibitory influence on purified hematopoietic cells, suggesting a possible mechanism of adiponectin action in culture. In contrast to lymphopoiesis, myelopoiesis was slightly enhanced in adiponectin-treated bone marrow cultures, and even when cultures were initiated with single lymphomyeloid progenitors. Finally, human B lymphopoiesis was also sensitive to adiponectin in stromal cell cocultures. These results suggest that adiponectin can negatively and selectively influence lymphopoiesis through induction of PG synthesis. They also indicate ways that adipocytes in bone marrow can contribute to regulation of blood cell formation.     

Suppression of the allograft response by implants of mature lymphoid tissues in larval Xenopus laevis

One hundred and twenty-two larvae of Xenopus laevis, the South African clawed toad, at developmental stages 48, 50, 52 and 54, were implanted in the tail with two allografts from adult tissues. In each case, one allograft was from kidney, while the other was either from kidney, thymus, spleen, or liver. In any particular host the two implants were always from the same donor and the implants were all visually matched in size. The experimental period was a maximum of nine days, so as to minimize the large numbers of changes normally accompanying larval progress from stage to stage. We are concerned with the timing of allograft response initiation under the implant conditions of each experimental group at a particular point in development. An allograft response was defined as an infiltration and accumulation of small lymphocytes in the “test” kidney allograft. Larvae of all stages developed allograft responses within one week post-implantation when the variable implant was from kidney, but implants from spleen and thymus suppressed both the timing of initiation and the subsequent intensity of the response. Spleen was more effective in this regard than thymus and both were more effective in the earlier larval stages. Liver proved to be toxic to the larvae. The relationship between the maturation of the lymphomyeloid tissues and external morphological staging is also discussed.     

The distribution of rapidly and slowly renewed T, B, and "null" lymphocytes in mouse bone marrow, thymus, lymph nodes, and spleen.

Combined radioautography and immunofluorescence were employed to discern the proportions of rapidly renewed (RR) and slowly renewed (SR) T, B, and null cells in mouse lymph nodes (LN), spleen (Spl), thymus (Thy), and the lymphocyte-rich fraction of bone marrow (BML). Thy and BML were found to consist predominantly of cells with a rapid turnover rate (95.4 and 76.9%, respectively) in accord with their roles as primary lymphoid organs. In contrast, the secondary lymphoid organs were primarily composed of SR cells (LN, 73.4%; Spl, 67.8%) and contained a more even admixture of the six lymphocyte subsets. Most cells in Thy were RR T cells (93.3%), whereas the predominant lymphocyte subpopulation in both LN and Spl consisted of SR T cells (56 and 50%, respectively), with RR T cells being much less frequent (14.6% LN; 6.8% Spl). BML contained both RR and SR cells which stained with the T-cell reagent (7.8 and 7.1%). These percentages are probably overestimates, however, since this reagent stained some nonlymphoid cells in BML. RR B cells were most plentiful in BML (31.4%) and Spl (23.3%), less common in LN (11.2%), and rare in Thy (0.4%). SR B cells were equally numerous in Spl (16%), LN (15.4%), and BML (15.3%). RR null cells were the most prevalent cell type in BML (37.7%), but were infrequent in other tissues (1.6% Thy, 2.1% LN, 2.1% Spl). SR null cells were rare in all tissues (0% Thy, 2% LN, 2.1% Spl, 0.7% BML). These experiments represent the first comprehensive investigation of the composition of the lymphomyeloid organs in terms of RR and SR T, B, and null cells. They conclusively demonstrate RR and SR lymphocytes in all three functional categories (T, B, and null) and show characteristic tissue distributions of the six lymphocyte subsets.     

The chicken's femoral-lymph nodules: T and B cells and the immune response.

Femoral lymph nodules (FLN), which are barely perceptible in normal birds, after a footpad injection of sheep red blood cells (SRBC) may either significantly enlarge (responder) or remain reduced in size (nonresponder). There were approximately 38% T cells and 53% B cells in the FLN of responder chickens. Significantly more plaque-forming cells (PFC) developed in the FLN than in the spleen after a footpad injection of SRBC. Total antibody, mercaptoethanol- (ME) resistant, and ME-sensitive fractions were significantly higher in birds given i.v. than in those given footpad injections. There were no differences in PFC and agglutinin titers between FLN-responders and nonresponders. The number of PFC in FLN exceeded the number of splenic PFC previously reported. The high PFC response of the FLN may reflect the large percentage of B cells in this lymphomyeloid tissue or the presence of antigen-experienced B cells in the FLN. Although FLN may influence a systemic immune response its major role appears to be restricted to a local response.