Some electrical properties of the surfaces of murine B- and T-lymphocytes and thymocytes. II. Effects of neuraminidase and ribonuclease.

The electrical properties of the peripheries of murine thymocytes, B-lymphocytes and T-lymphocytes were studied by measurement of electrophoretic mobilities and electron microscopic quantitation of adsorbed, positively charged CIH particles. The effects of neuraminidase and/or ribonuclease treatment upon these parameters were examined. Neuraminidase-susceptible groups accounted for 17%, 13% and 21% of the net surface negativity of T-lymphocytes, B-lymphocytes and thymocytes, respectively, and 28%, 63% and 78% respectively of particle binding. The calculated numbers of charges at the cellular electrokinetic surface per observed CIH particle were similar in control T-cells and thymocytes and higher than in B-lymphocytes. In neuraminidase-treated T- and B-lymphocytes the calculated charges per CIH particle were much lower than in thymocytes. These results may well indicate heterogeneities in the distribution of groups susceptible to neuraminidase, and also in the distribution and/or chemical nature of anionic groups susceptible to neither neuraminidase nor ribonuclease at the peripheries of different murine lymphocyte populations; however, at present we cannot discriminate between these and other possibilities.     

Colloidal iron hydroxide-binding to the surfaces of chick embryo fibroblasts transformed by wild-type and a temperature-sensitive mutant of Rous sarcoma virus.

The densities of colloidal iron hydroxide (CIH) particles binding to the surfaces of chick embryo fibroblasts were determined before and after transformation with wild type Rous sarcoma virus and a temperature sensitive (ts) mutant of this virus. On the basis of in vitro behavior, cells transformed by the ts virus manifest a malignant phenotype at 36 degrees C (permissive temperature) and appear normal at 41 degrees C (non-permissive temperature). At the permissive temperatures there is a significant increase in CIH particle-binding to spaces of cell surface between microvilli on the wild type and ts transformed cells. At the non-permissive temperature this significant increase in binding is only observed on the wild type transformant, while the density found on the ts transformant is not significantly different from the untransformed state. Therefore, in vitro characteristics of normalcy and malignancy are reflected in changes in the CIH binding properties of the cell surface spaces between microvilli. The CIH densities observed on the microvilli are significantly different from the density on the spaces between them for each of the classes of cells studied at either temperature. The microvilli are found to bind a lower density of particles in five of the six cases. No correlations between microvilli particle density and transformation to in vitro malignant characteristics were observed.     

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.     

The pool of free purine and pyrimidine nucleosides and bases in the thymocytes, and splenic T- and B-lymphocytes of C3HA mice during the growth of solid hepatoma 22a

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.     

Epigenome-wide profiling identifies significant differences in DNA methylation between matched-pairs of T- and B-lymphocytes from healthy individuals

Multiple reports now describe changes to the DNA methylome in rheumatoid arthritis and in many cases have analyzed methylation in mixed cell populations from whole blood. However, these approaches may preclude the identification of cell type-specific methylation, which may subsequently bias identification of disease-specific changes. To address this possibility, we conducted genome-wide DNA methylation profiling using HumanMethylation450 BeadChips to identify differences within matched pairs of T-lymphocytes and B-lymphocytes isolated from the peripheral blood of 10 healthy females. Array data were processed and differential methylation identified using NIMBL software. Validation of array data was performed by bisulfite pyrosequencing. Genome-wide DNA methylation was initially determined by analysis of LINE-1 sequences and was higher in B-lymphocytes than matched T-lymphocytes (69.8% vs. 65.2%, P ≤ 0.01). Pairwise analysis identified 679 CpGs, representing 250 genes, which were differentially methylated between T-lymphocytes and B-lymphocytes. The majority of sites (76.6%) were hypermethylated in B-lymphocytes. Pyrosequencing of selected candidates confirmed the array data in all cases. Hierarchical clustering revealed perfect segregation of samples into two distinct clusters based on cell type. Differentially methylated genes showed enrichment for biological functions/pathways associated with leukocytes and T-lymphocytes. Our work for the first time shows that T-lymphocytes and B-lymphocytes possess intrinsic differences in DNA methylation within a restricted set of functionally related genes. These data provide a foundation for investigating DNA methylation in diseases in which these cell types play important and distinct roles.     

Evidence for the recycling nature of the fibronectin receptor of macrophages

Plasma fibronectin (pFN) has been shown to mediate phagocytosis of several types of artificial particles and tissue debris by macrophages. In the present investigation some of the dynamic aspects of this receptor-mediated cellular process have been studied. Plasma fibronectin did not bind specifically to fibronectin (FN)-receptors of rat peritoneal macrophages at either 4 degrees C or 37 degrees C. On the other hand, pFN aggregated on the surface of gelatin-coated latex beads (gLtx) and 125I-labeled pFN covalently coupled to latex beads (pFN-Ltx) bound strongly to macrophages at both temperatures. Both of these particles were also internalized at 37 degrees C. Treatment of macrophages by chymotrypsin, thermolysin, or trypsin in a protein-free tissue culture medium did not affect either of the above reactions; however, pronase treatment strongly reduced both the binding and internalization of the pFN-coated particles. The pronase-treated macrophage monolayers in time regained their ability to bind and internalize pFN-gLtx when incubated in fresh tissue culture medium. Such recovery, however, did not take place when the medium contained cycloheximide. On the other hand, phagocytosis of pFN-gLtx was not affected directly by cycloheximide with untreated macrophages; this suggests that the FN-receptor recycles during sustained phagocytosis. This assumption was substantiated by the observations that some of the established lysosomotropic amines--i.e., chloroquine, dansylcadaverine, and dimethyldansylcadaverine--caused total inhibition of internalization without affecting the binding of particles to macrophages. Furthermore, chloroquine protected the FN-receptors against destruction by pronase. Together these results suggest that macrophage receptors for FN are protein, present both on the cell surface and intracellularly, and recycle between the plasma membrane and intracellular sites during phagocytosis.     

The binding of cationized ferritin at the surfaces of ehrlich ascites tumor cells: the effect of pH and glutaraldehyde fixation.

The densities of cationized ferritin (CF) particles binding to the surfaces of cultured Ehrlich ascites tumor cells were determined at pH 7.4, where the ferritin stain was applied either prior to or following glutaraldehyde fixation. The densities were also determined with CF adjusted to pH 1.9 and applied after fixation. For all fixed samples there was a higher density of particles bound to microvilli than to the spaces between them. Treatment with neuraminidase removed more particles from microvilli than from the inter-microvillus spaces, but did not reduce the levels of binding to the same value. When cationized ferritin is applied prior to fixation, an aggregation of the CF particles at the cell surface was observed, with the internalization of some clusters. This effect was independent of neuraminidase treatment.     

Mechanism of the adjuvant action: effect on the stem cell and B-lymphocyte migration from the bone marrow]

The authors studied the influence of synthetic polyelectrolytes of polyacrylic acid and poly-2-methyl-5-vinylpyridine and of complete Freund's adjuvant on the migration of stem cells and B-lymphocytes from mouse bone marrow. The stem cell count was evaluated by the number of splenic colonies; as to B-cell migration--it was assessed by the accumulation of the antibody-forming cells forming from B-lymphocytes migrating in the spleen in the transfer of fixed number of T-lymphocytes. As revealed the synthetic substances under study intensified the migration of stem cells and of B-lymphocytes to a much greater extent than Freund's adjuvant. The mechanisms of the influence of the adjuvants used on cell migration processes are discussed.     

Differences in the capacity of HLA sera to react with T- and B-lymphocyte populations]

A study was made of perculiarities attending the reaction of the HLA-sera with the T- and B-lymphocytes isolated from human blood. Lymphocytes were separated by removal of one of the cell subpopulation. T-lymphocytes were separated by the method of rosette-formation with sheep erythrocytes, with subsequent gradient density centrifugation. B-lymphocytes were separated similarly with the aid of rosette-formation with allogenous Rh-positive erythrocytes sensitized with Rh-sera with incomplete antibodies, and also sorption of B-lymphocytes on synthetic fiber. The cytotoxic activity of HLA-sera decreased after the removal of B-cells. But removal of T-lymphocytes was not accompanied by any reduction in the lymphocytotoxic activity. It is suggested that B-lymphocytes contained on their surface more HLA determinants than T-lymphocytes.     

Role of B-lymphocytes in inactivating allogenic stem cells]

Antisera to membrane antigens of B lumphocytes eliminated the capacity of lymphocytes to inactivate allogenous stem cells by 60%; however, lymphocytes from the lymph nodes of B mice possessed no inactivating capacity. T-lymphocytes were the main criteria inactivating allogenous stem cells. Cooperating with T-lymphocytes, B-lymphocytes probably contributed to inactivation of precursor cells realized by T-lymphocytes. However, the presence of B-lymphocytes in the killer cells population was not a determinant, since T-lymphocytes were capable of inactivating allogenous stem cells without any participation of B-lymphocytes.     

Expression of Thomsen-Friedenreich (TF) antigens on lymphocytes. I. Distribution of cryptic and exposed TF antigens on murine lymphocytes from different lymphoid organs: detection with an anti-TF monoclonal antibody and peanut agglutinin

Cryptic Thomsen-Friedenreich (TF) antigens were detected by the lectin peanut agglutinin (PNA) on the surface of murine lymphocytes after treatment of cells with neuraminidase. Thereby, a particular TF antigen could be distinguished using a monoclonal anti-TF antibody 49H8. In contrast to the known general galactoside specificity of PNA, the mAb was restricted to Gal beta(1-3)GalNAc/GlcNAc. Preincubation of cells with PNA abolished mAb 49H8 binding completely. However, only the intensity of staining with PNA was reduced by prior incubation of cells with the mAb. Cryptic TF antigens detected by the mAb were expressed on 39% of murine bone marrow cells, 88% of thymocytes, 62% of lymph node cells, and 65% of spleen cells. On the other hand, over 80% of the lymphatic cells carried cryptic PNA binding sites independent of the lymphoid organ they derived. In the thymus, a subpopulation of cells (76%) could be detected by PNA without neuraminidase treatment. Twenty-eight percent of thymocytes carried exposed mAb binding sites, too. All of them were shown to express further binding sites for PNA constantly. Therefore, a subpopulation of PNA-reactive, immature thymocytes can be distinguished by the mAb 49H8. During activation of splenic lymphocytes with PHA, the lymphoblasts completely lost their cryptic mAb binding sites while PNA reactivity was not affected. We conclude that the anti-TF mAb recognizes a particular TF antigen exposed on thymocytes and present in a cryptic form on other lymphocytes. The number of cells carrying mAb 49H8 binding sites varied, dependent on the organ from which the lymphocytes derived. PNA-reactive lymphocytes are distributed homogeneously in the lymphoid organs.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Immunoelectron microscopic localization of cell surface antigens on rat hepatocytes detected with monoclonal antibodies (HAM2 and HAM4).

The localization of surface antigens and the binding activity of two monoclonal antibodies, HAM2 and HAM4, which recognize the rat major histocompatibility complex (MHC) antigen class I and the rat hepato-renal antigen respectively, on dissociated (free) hepatocytes was examined by light (LM) and electron microscopy (EM), and by radioimmunoassay (RIA). Fixed hepatocytes, fixed before dissociation, and fresh hepatocytes, dissociated by collagenase, were treated by direct staining with HAM2- or HAM4-immunogold complexes (HAM2-gold and HAM4-gold). Some of the directly stained hepatocytes were further mixed with antimouse IgG-gold complex (IgG-gold) to supplement the direct staining. The polarity of the sinusoidal and contiguous faces and the bile canaliculus, i.e. the in situ morphology, was well preserved in the fixed hepatocytes, while the fresh cells had lost the polarity and were round. On the fixed hepatocytes HAM2-gold particles were distributed predominantly on the sinusoidal face, while HAM4-gold particles were localized on both the bile canalicular and sinusoidal faces. No different antigen distribution on the fresh cells was detected with the two antibodies. Supplementation by IgG-gold was noticeable in most cases. The extent of binding activity in both the immunogold and RIA experiments was lower in the fixed cells than in fresh cells. These results suggest that HAM2 and HAM4 are useful monoclonal antibodies for detecting the localization of the MHC class I antigen and the hepato-renal antigen on the hepatocytes, respectively.     

Effect of hyperbaric oxygenation on the course of experimental staphylococcal infection and on the immune status of the animal body

In guinea pigs infected with staphylococci by subcutaneous injection a decreased content of T-lymphocytes, an increased number of B-lymphocytes and lower levels of lysozyme and complement were observed. When subjected to the action of hyperbaric oxygenation, the animals, both intact or infected with staphylococci, showed the aggravation of staphylococcal infection, a decrease in the number of T-lymphocytes and an increase in the content of B-lymphocytes. In the intact animals hyperbaric oxygenation stimulated the production of complement and lysozyme, produced a decrease in the number of T-lymphocytes and an increase in the number of B-lymphocytes.     

Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr. The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique. On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically. One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume. The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol. Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane. Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.     

The effect of extracorporeal blood radiotherapy on the peripheral blood T and B-lymphocytes in chronic lymphadenitis]

B-lymphocytes and T-lymphocytes were determined before and after therapy in 12 patients with chronic lymphatic leukemia (CLL) treated with extracorporal blood radiation (ECIB). There was a significant decrease of B-lymphocytes (p less than 0.001) from 85.3 +/- 6.55% to 71.55 +/- 10.60% by ECIB, whereas no significant changes could be found in T-lymphocytes. 12 untreated CLL patients, whose B-lymphocytes amounted 83.25 +/- 6.79% with 7.5 +/- 3.42% of T-lymphocytes, were examined as group of comparison. From these findings ECIB is concluded to cause a decrease of accumulated B-cells.     

Localization and quantitation of anionic charge sites in fetal and neonatal alveolar basement membranes

A method utilizing biopsy sized samples of lung for anionic charge site localization in alveolar and capillary basement membranes in human tissue is discussed. Tissue fixed in either paraformaldehyde-lysine-periodate or 1% paraformaldehyde with 0.05% glutaraldehyde, cut into 30 mu sections, and incubated with the cationic probe, polyethyleneimine, was processed for electron microscopic analysis using standard techniques. Anionic charge sites were identified and regularly distributed in increments of approximately 40-50 nm in the lamina rara externa of the alveolar basement membrane, with lesser amounts found in the lamina rara interna and lamina densa. Anionic charge sites were also demonstrated in the interstitial portion of the capillary basement membrane and on cell surfaces. These methods can be used to more broadly define the localization of anionic charge sites in human lung tissue in both normal and pathologic states.     

Immunohistochemical identification of T- and B-lymphocytes delineated by the unlabelled antibody enzyme method

Summary T-lymphocytes and B-lymphocytes are identified in tissue sections of human tonsils by applying the unlabelled antibody enzyme method. The epithelium of the tonsils contains a majority of immunoglobulin-positive cells and fewer T-lymphocytes. In the subepithelial zones, areas composed of B-cells predominate, however, regions containing T-lymphocytes are also present. The latter are mainly arranged in the lamina propria around high-endothelial venules and often include plasma cells containing immunoglobulin. Follicles containing germinal centres display a complex structure which changes during development. The lymphocytic cap consists of densely packed lymphocytes, labelled heavily by anti-IgM and anti-IGD, and of individual T-lymphocytes. Germinal centres show a framework of immunoglobulin-positive dendritic reticular cells; they contain some heavily labelled lymphoid cells and several cells weakly labelled by anti-IgM and anti-IgA, as well as a small number of T-lymphocytes. Furthermore, the total areas of T- and B-lymphocytes measured by planimetry may differ considerably between different tonsils. Especially total areas of germinal centres vary to a great extent. The quantitative data on amounts of T- and B-cells achieved by planimetry are comparable to those reported in cellular suspensions of tonsils.     

Presence of glucocorticoid receptors in cultured thymic epithelial cells

We investigated the presence of glucocorticoid receptors (GC) in human thymic epithelial cells grown in primary cultures and in a pure epithelial rat cell line. These GR levels were compared to those determined concomitantly in fresh human thymocytes. The average number of sites were 54,457/cell for males (n = 8) and 58,224/cell for females (n = 8) with mean Kd values of 1.5 and 1.7 X 10(-8) M, respectively, in cultured human epithelial cells. These results are comparable to those obtained for rat thymic epithelial cells. Competition experiments showed that the relative affinities of the steroids tested were in decreasing order: dexamethasone greater than progesterone greater than testosterone and estradiol. This observation is compatible with binding to physiological GR. Moreover, the mean GR value appeared to be approximately 10 times higher for human thymic epithelial cells than for thymocytes. Thus, human epithelial cells as well as thymocytes should be considered as a specific target for glucocorticoid hormones.