Lysosomes in lymphoid tissue. II. Intracellular distribution of acid hydrolases

Differential centrifugation and density gradient isopycnic centrifugation have been used to fractionate homogenates of rat spleen and, in a few experiments, of rat thymus and cervical lymph nodes. The fractions have been analyzed for proteins, DNA, RNA, cytochrome oxidase, esterase, and up to 11 acid hydrolases. The results obtained indicate that the hydrolases are associated, at least largely, with cytoplasmic particles of lysosomal nature, and suggest further that these particles belong to two, and possibly three, distinct populations, perhaps reflecting the cellular heterogeneity of the tissues. The populations are identified as: (a) the L(19) population, the most important group, containing all 12 hydrolases and characterized by a modal density of about 1.19 in a sucrose-0.2 M KCl gradient; (b) the L(15) population with a modal density of 1.15, a group of apparently incomplete lysosomes containing cathepsin D and a few other enzymes, but very poor in, or entirely devoid of, several acid hydrolases, including cathepsins B and C; (c) the L(30) population, comprising all 12 enzymes and banding together with the nuclei at a density of 1.30 or higher. Lack of success in separating the latter group from the nuclei renders its significance unclear.     

Investigation of the dynamics of the spread of human immunodeficiency virus to brain and other tissues by evolutionary analysis of sequences from the p17gag and env genes.

The time of spread of human immunodeficiency virus type 1 (HIV-1) from lymphoid to nonlymphoid tissues in the course of infection was investigated by sequence comparisons of variants infecting a range of lymphoid and nonlymphoid tissues from three individuals with AIDS in the pl7gag gene and regions flanking the V1/V2 hypervariable regions. Phylogenetic analysis in both regions revealed several lineages in each individual that contained sequences from both lymphoid and nonlymphoid tissues such as the brain. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. Although individual pairwise comparisons of relatively short sequences such as p17gag are subject to considerable stochastic error, we found that the diversity of gag sequences in variants from lymphoid tissue was consistently lower than that found among variants amplified from the brain. By estimating mean synonymous pairwise distances in the p17gag region, we were able to make an approximate calculation of the ages of populations in different tissues. Those from lymphoid tissue ranged from 2.65 to 5.6 years in the three study subjects, compared with 4.1 to 6.2 years for variants in the brain. Indeed, variants infecting the brain were no more closely related to each other than they were to variants infecting other tissues in the body. In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This study is the first in which it was possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into nonlymphoid tissues and its possible adaptation for replication in different cell types.     

Role of dextran-specific suppressor T cells in the regulation of the immune response by a reactive form of dextran

Reactive forms of antigens or haptens have been shown to induce a state of hyporesponsiveness mediated in part by suppressor T cells. Injection of Balb/c x C57B16 F1 (CB6F1) mice with a reactive form of dextran B1355S (periodate oxidized dextran, dex-P) specifically reduced responses to dextran immunization within 1 day after dex-P treatment. This unresponsiveness lasted at least 23 days and required a reactive form of dextran for its induction since native dextran and oxidized/reduced dextran failed to induce tolerance. Furthermore, hyporesponsiveness could be induced by iv injection of dextran-coupled cells, especially peripheral blood lymphocytes, a result which suggests that in vivo coupling to cellular antigens is involved in dex-P-induced hyporesponsiveness. Suppression of the anti-dextran response could be transferred to normal mice with T-cell-enriched spleen cell populations from dex-P-injected mice. Interestingly, the presence of B cells in the transferred cell preparations interfered with detection of suppression. Both Lyt 1+2- and Lyt 1-2+ cells were involved in the dex-P-induced suppression; indeed, mixtures of these types of T cells led to the most profound degree of suppression. The suppressive activity of spleen cells from dex-P-injected mice could be removed by passage over dextran-coated plates. Moreover, cells eluted from the plates specifically suppressed anti-dextran responses of normal mice, indicating that dex-P injection induces a population of antigen-binding suppressor cells. This system will allow the study of the suppressor-T-cell receptors in a well-defined idiotypic system.     

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45⁺ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45⁺ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.     

Decreased ganglioside neuraminidase activity in fibroblasts from mucopolysaccharidosis patients. Inhibition of the activity in vitro by sulfated glycosaminoglycans and other compounds

The neuraminidase activities towards the ganglioside substrates GD1a, GD3 and GM3 were found to be markedly diminished in homogenates of fibroblasts cultured from patients with various genetic mucopolysaccharidoses. Mixing normal and patients' fibroblast homogenates revealed this effect to be due to the presence of diffusible inhibitors. The neuraminidase acting on the trisaccharide sialyllactose, on the other hand, showed normal activity in all the cell lines tested. Experiments in vitro revealed the sulfated glycosaminoglycans chondroitin 4-sulfate and heparin, the polysaccharide dextran sulfate, and the trypanocidal drug suramin to be strongly inhibitory on the ganglioside GD1a neuraminidase activity of normal fibroblast homogenates. Regarding chondroitin 4-sulfate, this inhibition was of the non-competitive type. A disulfated tetrasaccharide prepared from chondroitin 4-sulfate, on the other hand, was not at all inhibitory. These and additional findings led us to propose a model for the interaction between enzyme and inhibitor, involving a 'clamping' mechanism by the polysulfated compounds. We conclude that the decreased ganglioside neuraminidase activities of mucopolysaccharidosis fibroblasts are due to an inhibition by the accumulated sulfated glycosaminoglycans and that such inhibition is responsible for the storage of certain gangliosides in the tissues of the patients.     

Characterization of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents.

The myelin-associated glycoprotein (MAG) can be obtained from adult mouse brain from detergent-lysates of a crude membrane fraction as a 96-100 kd form (detergent solubilized MAG), and from 100,000 g supernatants of homogenates as a 90-96 kd form (soluble MAG). The soluble form distributes into the Triton X-114-poor aqueous phase, while detergent-solubilized MAG predominantly enters the Triton X-114-rich phase. Both molecular forms bind to heparin in hypo- and isotonic buffers. Soluble MAG binds to several collagens (type G, I, II, III, IV, V, VI, IX) with a kd of 5.7 X 10(-8) M for collagen type IX and 2.0 X 10(-7) for collagen type IV. Binding of 125I-labeled MAG to collagen G can be completely inhibited by unlabeled MAG and collagen G, but not by heat-denatured collagen. MAG does not bind to itself, laminin, fibronectin, or the neural cell adhesion molecules L1 and N-CAM. Binding of MAG to collagen G is most effectively blocked by a high molecular weight dextran sulfate, heparan sulfate and heparin, with chondroitin sulfate and a low molecular weight dextran sulfate being less potent blockers. These findings are in agreement with previous observations on the localization of MAG in basal lamina and interstitial collagens of the sciatic nerve in situ.     

Thymus-derived lymphocyte differentiation and lymphocytic leukemias. I. Evidence for the existence of functionally different subpopulations of thymus-derived cells in leukemic AKR mice

The effects of transplanting thymic (LTC), splenic (LSC), and lymph node (LLNC) lymphocytes derived from overtly leukemic AKR mice into preleukemic syngeneic animals were studied. Each of these thymus-derived (T cell) populations produced a different and distinct pathology in recipient mice. Animals receiving LTC exhibited thymoma and enlargement of peripheral lymphoid tissues. Gross organomegaly was also noted in mice given LSC, but thymic atrophy was uniformly observed. The thymus appeared normal in mice receiving LLNC, but marked enlargement of peripheral lymphoid tissues again were observed. The differences noted in disease pathologies correlated with the “homing” patterns of the subpopulations investigated. These findings suggest that subpopulations of T cells exist in mice with a thymus-derived neoplastic disorder.     

Suppressor cells in homograft tolerant rats.

If sufficient normal syngeneic lymphocytes to effect skin graft rejection are transferred to homograft tolerant rats, a prolonged period elapses before lymphoid cells from the recipient acquire normal levels of GvH responsiveness against tissues of which the donor was previously tolerant (Silvers and Billingham, 1970; Elkins, 1972; Miyamoto and McCullagh, 1974). Although the ability of lymphoid populations of such animals to mount GvH reactions can be demonstrated to reside in donor type cells during the weeks immediately after transfer, reactive cells are ultimately derived from the host itself (Elkins, 1973; Miyamoto and McCullagh, 1974). Not only are lymphoid cells from tolerant rats which have been injected recently with normal lymphocytes poorly responsive in a GvH assay, but they have been observed in some experiments to suppress the GvH activity of normal syngeneic lymphoid cells (Elkins, 1972; Atkins and Ford, 1972). It is not clear whether the cells mediating suppression of the normal lymphocytes were derived from the tolerant host itself or, alternatively, from the normal lymphocytes injected into it to terminate the tolerant state. The present experiments sought to delineate the origin of any suppressor cells within populations of lymphocytes collected from rats in which tolerance had recently been terminated. The indicate that suppression of the normal donor cells within such populations may be exerted by cells derived from the tolerant host.     

Human BDCA-1-positive blood dendritic cells differentiate into phenotypically distinct immature and mature populations in the absence of exogenous maturational stimuli: differentiation failure in HIV infection

Current immunological opinion holds that myeloid dendritic cell (mDC) precursors migrate from the blood to the tissues, where they differentiate into immature dermal- and Langerhans-type dendritic cells (DC). Tissue DC require appropriate signals from pathogens or inflammatory cytokines to mature and migrate to secondary lymphoid tissue. We show that purified blood mDC cultured in vitro with GM-CSF and IL-4, but in the absence of added exogenous maturation stimuli, rapidly differentiate into two maturational and phenotypically distinct populations. The major population resembles immature dermal DC, being positive for CD11b, CD1a, and DC-specific ICAM-3-grabbing nonintegrin. They express moderate levels of MHC class II and low levels of costimulatory molecules. The second population is CD11b(-/low) and lacks CD1a and DC-specific ICAM-3-grabbing nonintegrin but expresses high levels of MHC class II and costimulatory molecules. Expression of CCR7 on the CD11b(-/low) population and absence on the CD11b(+) cells further supports the view that these cells are mature and immature, respectively. Differentiation into mature and immature populations was not blocked by polymyxin B, an inhibitor of LPS. Neither population labeled for Langerin, E-cadherin, or CCR6 molecules expressed by Langerhans cells. Stimulation of 48-h cultured DC with LPS, CD40L, or poly(I:C) caused little increase in MHC or costimulatory molecule expression in the CD11b(-/low) DC but caused up-regulated expression in the CD11b(+) cells. In HIV-infected individuals, there was a marked decrease in the viability of cultured blood mDC, a failure to differentiate into the two populations described for normal donors, and an impaired ability to stimulate T cell proliferation.     

Effects of estrogen on the lymphoid regeneration and immune response in irradiated and marrow-reconstituted mice.

Various doses of estriol (E3) were given to mice intraperitoneally, immediately after lethal irradiation and marrow reconstitution. The assessment of the plaque-forming cell (PFC) response to sheep erythrocytes in the spleen and the histological assessment of lymphoid tissues were carried out 30 days later. The effects appeared to be dose-dependent and resulted in a marked suppression of the PFC response. The depletion of lymphocytes was dramatic and dose-dependent in the thymus, and in the thymus-dependent and in the thymus independent areas of the peripheral lymphoid tissues. These results suggest that E3 acts on the differentiation of stem or precursor cells toweard both the populations of T and B lymphocytes. Although E3, given on day 7 after irradiation and marrow reconstitution, suppressed the lymphoid regeneration and PFC response markedly, E3 given on day 14 had no effect. On day 7 the majority of regenerating lymphoid tissues were large pyroninophilic cells and on day 14, small lymphocytes. These results suggest that the precursor or immature lymphocytes are sensitive to E3, while mature lymphocytes are resistant. Lymphoid regeneration and PFC response were retarded in mice irradiated and reconstituted with bone marrow cells from donors pretreated with E3. These results suggest that E3 acts on the stem or precursor cells capable to differentiate in the direction of lymphoid populations and reduce their number in the bone marrow.     

重金属污染下曼陀罗种群分化的RAPD分析

将不同空间地段上获得的同一种质但污染年代各不相同的4个曼陀罗材料种子易地种植在同一模拟重金属污染生境中,对这4个曼陀罗种群进行RAPD分析,结果表明,在105个检测位点中发现有78个位点呈多态性。在这些多态位点中未发现与重金属抗性有关的特异性多态DNA片段。S hannon-Weiner指数计算结果表明,在短期污染时间内曼陀罗种群遗传多样性水平降低。随着污染时间的推移,曼陀罗种群逐渐在污染迹地上稳定下来,曼陀罗种群遗传多样性水平有所回升和提高,4个曼陀罗种群遗传多样性由高到低排列顺序为L＞CK＞M＞S。遗传多样性指数表明曼陀罗种群间变异程度远小于种群内的遗传变异。4个种群两两间遗传距离较小,遗传距离最大的种群为L和S,最小的为L和CK种群。因此,在重金属胁迫环境选择下,曼陀罗种群发生了一定程度的分化与微进化,轻高水平的遗传多样性可能是植物适应重金属污染胁迫环境的基础。     

Lysosomes in lymphoid tissue. I. The measurement of hydrolytic activities in whole homogenates

Methods have been developed for the quantitative assay of cytochrome oxidase, esterase, and 11 acid hydrolases in rat-spleen homogenates. These methods seem to be applicable also to other lymphoid tissues. Preliminary studies, extended to nine of the acid hydrolases, indicate that these enzymes occur in partly latent and sedimentable form and that they can be unmasked and rendered soluble by some of the treatments that liberate the enzymes from rat-liver lysosomes. The spleen particles appear to be very sensitive to mechanical injury, a property which necessitates special precautions in homogenizing the tissue. Agglutination of spleen particles takes place to a larger extent in 0.25 M sucrose than in 0.15 M KCl.     

Characterization of T lymphocyte colony-forming cells in the mouse. The colony-forming cell is confined to Lyt-1,2,3+ cell subsets in the thymus and the lymph nodes

When cell populations from the thymus were studied with FACS, it was found consistently that the brightly labeled Thy-1.2+ populations contained very few T colony-forming cells (CFC), while these latter cells were numerous in the cell populations showing lower Thy-1.2 antigen density. This was paralleled by findings after peanut agglutinin (PNA) separation that showed enrichment of CFC in the PNA-negative medullary population, and by sorting based on TL, T-200, and H-2 determinants or light scatter properties of the cells. By FACS sorting of Lyt-labeled thymic cells, it was also shown that CFC were predominantly present in cell populations that were brightly Lyt-1+, and exclusively in populations that were Lyt-2+ and Lyt-3+. After FACS sorting of lymph node cells, no major differences in colony formation were found between dully- and brightly-labeled Thy-1.2+ or Lyt-1+ populations, or between lymphoid cells showing different light scatter characteristics. In addition, it was shown that CFC--like thymic CFC--were of the Lyt-1,2,3+ phenotype. It is concluded that the CFC may be present in several differentiation steps of Lyt-1,2,3+ cell lines, and that the frequency of these cells increases from the thymic cortex via the medulla and to peripheral lymphoid tissues.     

Regulation of the anti-alpha (1,3) dextran response: two populations of dextran-reactive B cells that differ in their T cell requirements for induction to antibody synthesis

The in vitro antibody response to dextran B1355S, a thymus-independent Type 2 antigen, requires T cell-derived lymphokines but is not thought to require an activation signal from an antigen-specific T helper cell. The present study demonstrates that there are two dextran-reactive B cell populations in BALB/c mice with respect to the T cell requirements for the generation of antibody-forming cells. One population found among dextran-reactive spleen B cells from 12- to 14-mo-old BALB/c mice generated anti-dextran PFC in the presence of B cell growth factor (BCGF II) and IL 2 or the combination of BCGF II, IL 2, and IFN-gamma. A second population of dextran-reactive B cells found in spleen and Peyer's patches of 2-mo-old unprimed mice did not respond to these same lymphokines, but did generate anti-dextran plaque-forming cells in the presence of Thy-1.2+, L3T4+ T cells from Peyer's patches. However, splenic B cells obtained from 2-mo-old mice that had been primed with dextran 2 to 3 days after birth were shown to be responsive to the same lymphokines as dextran-reactive B cells from 12- to 14-mo-old mice. These results suggest that previous priming with dextran B1355S induces a dextran-specific B cell population that can be activated to antibody-forming cells in the presence of antigen and T cell-derived lymphokines, whereas a second, unprimed population requires an additional activation signal from L3T4+ T cells.     

Spontaneous plaque-forming cells against autologous erythrocytes develop in cultures of normal rabbit appendix cells.

Cultured appendix and, to a lesser extent, mesenteric lymph node cells from normal, unimmunized rabbits spontaneously develop PFC against several erythrocyte species, including sheep erythrocytes (SRBC), trypsin-treated, autologous erythrocytes (TRRBC), and, most importantly, untreated, autologous erythocytes (RRBC). Cells from most other lymphoid tissues of the rabbit, including the spleen, fail to develop spontaneous, anti-autologous PFC in culture. This failure seems to be due to a lack of appropriate precursors among the cells comprising their populations, rather than to an inhibition by some suppressor cell population. The development of spontaneous PFC in vitro, and their virtual absence among appendix cells freshly removed from the rabbit implies an effective regulation on their expression in situ. This regulation may involve, in part, antigen itself. The development of the anti-autologous RRBC specificities may be a consequence of the intimate association of the gut-associated lymphoid tissue with the rich antigenic milieu in the intestinal lumen, part of which may present antigens cross-reactive with self antigens.     

Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells

We have recently demonstrated the presence of three populations of dendritic cells (DC) in the murine Peyer's patch. CD11b(+)/CD8alpha(-) (myeloid) DCs are localized in the subepithelial dome, CD11b(-)/CD8alpha(+) (lymphoid) DCs in the interfollicular regions, and CD11b(-)/CD8alpha(-) (double-negative; DN) DCs at both sites. We now describe the presence of a novel population of intraepithelial DN DCs within the follicle-associated epithelium and demonstrate a predominance of DN DCs only in mucosal lymphoid tissues. Furthermore, we demonstrate that all DC subpopulations maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of cytokines upon exposure to T cell and microbial stimuli. Only myeloid DCs from the PP produce high levels of IL-10 upon stimulation with soluble CD40 ligand(-) trimer, or Staphylococcus aureus and IFN-gamma. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12p70 following microbial stimulation, whereas no DC subset produces IL-12p70 in response to CD40 ligand trimer. Finally, we show that myeloid DCs from the PP are particularly capable of priming naive T cells to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, while lymphoid and DN DCs from all tissues prime for IFN-gamma production. These findings thus suggest that DC subsets within mucosal tissues have unique immune inductive capacities.     

Temporal stability in size distributions and growth rates of three Esox lucius L. populations. A result of cannibalism?

The population total length ( L _T) structures and individual growth trajectories for three stream living pike Esox lucius populations were studied for 7 years. All three populations exhibited small variation in both population L _T structure and individual growth trajectories over time. These dynamics contrasted to the much more variable population L _T structure of perch Perca fluviatilis studied previously. The difference in population dynamics between the two species was related to differences in prey:predator size ratios. The pike populations in the more open and larger streams grew to larger sizes, but this difference in life history did not affect population dynamics of pike. It is concluded that (1) cannibalistic population dynamics may be predicted from individual life-history characteristics such as minimum and maximum victim:cannibal size ratios and (2) the cannibal-driven population dynamics observed in pike seems to be robust to variation in environmental conditions (system openness).     

Quantitative study of regression and regeneration of the thymic cell population after X-irradiation in the newtPleurodeles waltlii Michah

Summary The changes in cell numbers of different thymic cell populations and the conditions governing the regeneration of these populations and the thymus itself were examined after X-irradiation (700 rads) of different parts of the body. The general effects of the irradiation were studied in each experimental group in terms of mortality and growth rate. The particular effects on each thymic cell population were studied by the measurement of mitotic activity and of evaluation of the changes in numbers among these populations in the thymus itself, and were compared with the effects in the granulopoietic layer of the liver and in the spleen. The great reduction in the number of lymphocytes after irradiation demonstrates that they are more radiosensitive than other cell types; this reduction can be compensated for by the arrival of new lymphoid cells originating from other lymphoid organs (if they have been protected from irradiation) and by allowing thymic regeneration. Thus, irradiation has indirect effects on non-irradiated areas, and demonstrates that the lymphoid cell population has a high potential for multidirectional migration.     

Synergy between subpopulations of normal mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity specific for "modified self" antigens.

Responding lymphoid cells cultured in vitro with irradiated trinotrophenyl (TNP)-modified syngeneic spleen cells develop direct cell-mediated cytotoxicity which is specific for target cells bearing both the TNP moiety and histocompatibility determinants of the modified sensitizing cell. Two subpopulations of normal mouse spleen cells have been shown to synergize in the in vitro generation of specific cell-mediated cytotoxicity to these "modified self" antigens. The synergizing populations are nylon wool column-adherent and column-nonadherent fractions of normal mouse spleen. When mixtures of these two cell populations are cultured in vitro with irradiated TNP-modified syngeneic spleen cells, greater cytotoxicity is generated in the two populations sensitized separately. The synergizing cell in the column-adherent population is resistant to lysis by rabbit anti-mouse brain serum, is distinct from the cytotoxic effector T lymphocyte, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by peritoneal cells. These results suggest that it is a non-T cell which may be distinct from the macrophage.