Selective adsorption of immune lymphocytes, synthesizing DNA, on corresponding target cells]

Lymphocytes of inbred mice immunized with allogenic tumour cells were labelled in vitro or in vivo by 3H-thymidine, washed out and incubated with target cells in the presence of "cold" thymidine. A fraction of labelled small and medium lymphocytes was demonstrated by autoradiography to be absorbed rapidly and specifically onto the corresponding target cells. When the non-labelled immune lymphocytes were preincubated with target cells for 2 hours, and 3H-thymidine was added to the medium at various time periods, the percentage of labelled small and medium lymphocytes which adhered to the target cells appeared to be reduced in the course of the incubation, with the target reaching the initial value after an 8-hour contact. Small and medium immune lymphocytes which synthesize DNA are supposed to be the effector killer fraction which do not transform into blasts during the interaction with the target cells.     

Expression of leucocyte antigens by cells from the metrial gland of the pregnant rat

Summary The function of the metrial gland of the rat, and particularly of its characteristic population of granulated cells, remains unknown. However, several lines of evidence suggest that the granulated cells may derive from lymphocytes, and play a role in the immunology of pregnancy. In this study, antigen expression by granulated and other cell populations from the metrial glands of rats at Days 13 and 14 of pregnancy was studied by an indirect immunoperoxidase method. Acetone-fixed frozen sections, and cytocentrifuge preparations of collagenase-dispersed metrial gland tissue in which numbers of granulated cells had been increased by density-gradient centrifugation, were used. The primary antibodies used recognised, inter alia, B lymphocytes (MRC OX-3, MRC OX-6, MRC OX-12), T lymphocytes (MRC OX-8, W3/25, MRC OX-19), neutrophils (MRC OX-42) and cells of the monocyte/macrophage series (MRC OX-3, MRC OX-6, MRC OX-42, MRC OX43). The majority of the granulated cells, including smaller, immature forms, were unlabelled by any of these antibodies. Some lymphocytes, and varying numbers of larger, non-granulated cells, were labelled by OX-6, OX-12, W3/ 25, OX-42 and OX-43. In addition to lymphocytes, labelled cells included neutrophils (OX-42), endothelial cells (OX-43), and probably some macrophages (OX-6, OX-43). OX-12, which recognises the kappa chain of rat IgG, labelled some large cells which may have been stromal cells. These findings do not support the concept that the granulated cells are derived from lymphocytes.     

Incapacitation of fibrosarcoma cell by polyclonally activated murine T cell.

A piece of lymph node containing polyclonally-activated lymphocytes when transplanted in the anterior eye chamber of mouse along with solid piece of fibrosarcoma from syngeneic Swiss mice, dramatically inhibited the tumour-induced-vasodilatation and neo-vascularization. If the tumour explants were incubated in vitro with activated lymphocytes prior to transplantation, such angiogenic reactions was significantly reduced. These explants were incapable of incorporating radioactive thymidine in vitro. Furthermore, the cytotoxic ability of activated lymphocytes towards 51Cr labelled tumour target cells was of significant level indicating the possible mechanism of immunological reactiveness of Con A-stimulated lymphocytes to 3'-methylcholanthrene-induced tumour cells of syngeneic origin.     

Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells

Vascular endothelial dysfunction is thought to play a prominent role in systemic anthrax pathogenesis. We examined the effect of anthrax lethal toxin (LTx), a key virulence factor of Bacillus anthracis, on the expression of vascular cell adhesion molecule-1 (VCAM-1) on normal and cytokine-stimulated human lung microvascular endothelial cells. Confluent endothelial monolayers were treated with lethal factor (LF), protective antigen (PA), or both (LTx) in the presence or absence of tumor necrosis factor-alpha (TNFalpha). LTx enhanced cytokine-induced VCAM-1 expression and monocyte adhesion. LTx alone had no effect on VCAM-1 expression. LF, PA or the combination of a catalytically inactive mutant LF and PA failed to enhance cytokine-induced VCAM-1 expression. Treatment with inhibitors of mitogen-activated protein kinase kinases (MEKs) and mitogen-activated protein kinases did not reproduce the VCAM-1 enhancement effect of LTx, a known MEK metalloprotease, suggesting LTx-mediated MEK cleavage may not be a contributing factor.     

Secretory processes in lymphocyte function

The secretion of immunoglobulin by plasma cells has been considered a classical example of the non-regulated pathway of protein secretion, in which newly synthesized protein is processed by the Golgi, packaged into small vesicles, and immediately secreted without intracellular storage. In the case of lymphokine secretion by T lymphocytes, it is generally not clear whether this non-regulated pathway is also being used, as opposed to the regulated pathway which has been proposed to operate in the cytotoxic lymphocyte mechanism. In this case, as in mast cells and endocrine cells, proteins are synthesized and then stored in cytoplasmic granules. The secretion is triggered (regulated) by a membrane receptor-ligand interaction, which for the cytotoxic lymphocytes is part of the target cell binding process. In cytotoxic T lymphocytes, this secretion process can be measured by following the appearance of a granule serine protease in the medium, and it has been shown to be triggered by target cells or by immobilized antibodies which bind the T celt receptor complex. In addition to cytotoxic lymphocytes, cloned T helper cells contain this serine protease in cytoplasmic granules with a low internal pH. Helper lymphocytes secrete this enzyme in response to (1) soluble antigen which has been processed by cells bearing the appropriate MHC antigens; (2) immobilized antibodies against the T cell receptor complex; (3) a combination of phorbol ester and calcium ionophore. Thus in both helper and cytotoxic lymphocytes, the regulated pathway of protein secretion clearly operates after triggering by the T cell antigen receptor.     

Higher tyrosine protein kinase activity in resting lymphocytes than in proliferating normal or leukaemic blood cells

We have studied tyrosine phosphorylation in particulate fractions from 11 leukaemic cell lines by using as substrate either a synthetic tyrosine containing peptide or the endogenous proteins. The results were compared with those obtained using similar fractions from normal lymphocytes and bone marrow cells. Particulate fractions from all the leukaemic cell lines and normal bone marrow cells exhibited lower levels of tyrosine protein kinase activity compared to normal lymphocytes. When the phosphorylation of endogenous substrates was assayed, proteins were phosphorylated on tyrosine residues (rather than serine or threonine residues) to a larger extent in normal lymphocytes than in leukaemic cell lines. Separation of labelled endogenous substrates on sodium dodecyl sulfate-polyacrylamide gels showed a number of phosphorylated alkali-resistant bands in the range 14-175kd in the lymphoid cell lines; normal lymphocytes exhibited a smaller number of strongly phosphorylated bands. Normal lymphocytes from different individuals showed reproducible patterns of phosphorylated substrates. Normal bone marrow cells and myeloid leukaemia lines showed weak, if any, phosphorylation. Among the leukaemic cell lines no particular pattern of phosphorylated substrates common to cells of similar phenotype could be detected. We suggest that the level of overall tyrosine protein kinase activity in these fractions reflects their position in the cell cycle rather than their normal or malignant status.     

THE MIGRATION OF LYMPHOCYTES ACROSS SPECIALIZED VASCULAR ENDOTHELIUM

The chain of lymph-nodes in the rat mesentery was isolated and the preparation was perfused via cannulae in the superior mesenteric vessels. The perfusate consisted of serum to which labelled lymphocytes had usually been added. The entry of radioactively labelled lymphocytes from the blood vessels into the lymph-nodes was studied by scintillation counting and autoradiography. It was observed that: (1) In the perfused node labelled lymphocytes localized in and around post-capillary venules in the paracortex as they do early after i.v. injection. (2) The number of lymphocytes which entered the node was directly proportional to the concentration in the perfusate over the range studied. The proportion of cells retained in the node varied considerably around a mean of 11 % of lymphocytes reaching it. (3) The isolated lymph-node released few if any lymphocytes into the effluent (venous) perfusate. (4) Large lymphocytes migrated into isolated lymph-nodes slightly more readily than did small lymphocytes. (5) Unlike intact cells isolated lymphocyte membranes did not adhere to specialized vascular endothelium.     

MIGRATION OF SMALL LYMPHOCYTES IN ADULT MICE DEMONSTRATED BY PARABIOSIS

Parabiotic BALB/C mice were used to study the traffic of small lymphocytes in immunological mature but unchallenged mice. By giving ³H-thymidine (³H-TdR) injections to only one member (A) of a pair by preventing the escape of the radioactive isotope to the other member (B), the kinetics of newly-formed cells was followed. Less than 10% labelled small lymphocytes were found in the peripheral lymphoid tissues of both A and B members, while the thymusses and bone marrows of A members showed labelling percentages up to 70% in this period. Hardly any labelled cells gained entrance into the thymus while a detectable number was found in the bone marrows of B members. Results from pairs set up to follow migration of long-lived lymphocytes revealed that labelled cells detected 4–5 weeks after injections were equilibrated between the peripheral tissues and the bone marrows of the partners. Very few labelled cells were seen in the thymic medulla and none were observed in the thymic cortex, germinal centres or medullary cords of lymph nodes from any B member. It was concluded that short-lived small lymphocytes are formed primarily in the thymus and bone marrow and the migration of these cells is limited in adult animals. Furthermore, the vast majority of long-lived small lymphocytes are freely recirculating, and these cells gain entrance to and are normal residents in the bone marrow.     

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.     

Action of delta 9-tetrahydrocannabinol on the pool of acid soluble nucleotides

The effects of delta 9-tetrahydrocannabinol (THC) treatment on acid soluble pools of uridine nucleoside and nucleotides were investigated in Tetrahymena pyriformis and in isolated mouse lymphocytes and spermatogenic cells. In THC treated Tetrahymena and mouse lymphocytes the uptake of labelled precursor into acid soluble pools of uridine nucleoside and nucleotides fluctuated, whereas in pachytene spermatocytes and round spermatid cells the labelled pool was reduced. The reduction in the labelled pool measured in mouse spermatogenic cells was attributed primarily to a reduction in radioactively labelled uridine nucleoside. Treatment of Tetrahymena in high concentrations of THC (960 and 3,200 microM) resulted in an increase of labelled uridine nucleoside and a reduction in the amount of labelled uridine nucleotides. Expansion of the acid soluble pool with radioactive uridine resulted in small differences in labelled nucleoside and nucleotides in control and THC treated Tetrahymena and mouse lymphocytes. The results are discussed in terms of the effects of THC on macromolecular synthesis in various cellular systems.     

The migration of lymphocytes across specialized vascular endothelium. I. The entry of lymphocytes into the isolated mesenteric lymph-node of the rat.

The chain of lymph-nodes in the rat mesentery was isolated and the preparation was perfused via cannulae in the superior mesenteric vessels. The perfusate consisted of serum to which labelled lymphocytes had usually been added. The entry of radioactively labelled lymphocytes from the blood vessels into the lymph-nodes was studied by scintillation counting and autoradiography. It was observed that: (1) In the perfused node labelled lymphocytes localized in and around post-capillary venules in the paracortex as they do early after i.v. injection. (2) The number of lymphocytes which entered the node was directly proportional to the concentration in the perfusate over the range studied. The proportion of cells retained in the node varied considerably around a mean of 11% of lymphocytes reaching it. (3) The isolated lymph-node released few if any lymphocytes into the effluent (venous) perfusate. (4) Large lymphocytes migrated into isolated lymph-nodes slightly more readliy than did small lymphocytes. (5) Unlike intact cells isolated lymphocyte membranes did not adhere to specialized vascular endothelium.     

A monoclonal antibody identifying a T-cell marker in the horse

A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23A precipitated a molecule of approximately 69 kDa from 125Iodine labelled horse lymphocytes.     

Reversible Inhibition of Lymphocyte-mediated Cytotoxicity by Cytochalasin B

ALLOGRAFT immunity is characterized by the appearance of sensitized lymphocytes which are specifically able in vitro to destroy target cells carrying the sensitizing alloantigens. These cytotoxic lymphocytes represent thymus-derived effector cells^1,2 and are quite distinct from alloantibody-producing cells, which are also formed during induction of allograft immunity³. Although contact between viable cytotoxic lymphocytes and target cells is necessary for destruction, the events which lead to target cell lysis are still unknown⁵.     

Studies on the specificity of in vitro induced lymphocytotoxicity to SV40-transformed fibroblasts.

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from AKR-mice (H-2k) and from BALB/c-mice (H-2d) to syngeneic SV40-transformed fibroblasts. The T cell-dependent cytotoxicity was specific for target cells expressing the same H2-specificity as the immunizing cells. Nontransformed fibroblasts as stimulator cells did not induce efficient cytotoxicity to transformed or nontransformed target cells. Incubation with phytohemagglutinin during the sensitization period modified the specificity of the T cell-mediated lysis of syngeneic SV40-transformed fibroblasts: allogeneic as well as syngeneic target cells were destroyed by these effector cells. However, the polyclonal stimulant activates preferentially cytotoxicity to H2-matched target cells. The in vitro generation of cytotoxic effector cells was restricted to living SV40-transformed fibroblasts as immunizing cells; it was not possible to immunize lymphocytes in the presence of membrane proteins prepared from the SV40-transformed cells. The cytotoxicity of the in vitro immunized lymphocytes was inhibited by incubation with membrane protein preparations from syngeneic or allogeneic SV40-transformed fibroblasts.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

Life Span,Distribution and Radiosensitivity of the Large Lymphocyte

ALTHOUGH little is known about the origin or functions of the larger lymphocytes in the blood, a relationship to the small lymphocytes has been inferred, chiefly from similarities in gross morphology, staining characteristics and from the fact that lymphocytes exhibit a range of sizes which makes a distinction between large and small cells seem arbitrary. We have been studying lymphocyte kinetics by establishing cross-circulation between pairs of syngeneic rats, in which the lymphocytes of one member had been labelled in vivo by repeated injections of tritiated thymidine (³H-TdR) and we found that some time after the period of labelling, most of the isotope in blood leucocytes resided in the large lymphocytes. This suggested a way of studying a class of lymphocyte which has received scant attention so far.     

Antibody-dependent cytolytically active human leukocytes: an analysis of inactivation following in vitro interaction with antibody-coated target cells.

A leukocyte population consisting of approximately 85% lymphocytes, prepared from human peripheral blood by centrifugation through a Ficoll-Hypaque gradient, was studied for its capacity to destroy antibody-coated human liver (Chang) cells in vitro. Cytolysis was a rapid event: increased ionic flux (86Rb) from the target cell occurred within 10 min of the addition of effector cells. Kinetic analysis of target cell destruction (51 Cr release) was compatible with a "one hit" hypothesis, thereby indicating that cytolysis resulted from a single collision was an effector cell. The initial rate of cytolysis was linear and related to the number of leukocytes added, but lysis at all of the leukocyte to target cell ratios tested ceased after 5 hr. The number of target cells killed at that time was directly proportional to the number of leukocytes added. While the lytic capacity of the effector population was totally depleted after incubation with antibody-coated target cells, cytotoxicity was not affected by co-culturing leukocytes with Chang cells treated with pre-immune serum. The cytotoxic effector cells functioning in this antibody-dependent lytic system are thus to be contrasted with killer T cells, whose lytic activity is not compromised by interaction with homologous target cells. It was estimated that approximately 4% of the leukocyte population employed could kill antibody-coated Chang cells, a figure consistent with the estimated frequency of "null" cells within human peripheral lymphocytes.     

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autoimmune encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4+ T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4+, CD8+, CD14+ (monocytes) and CD56+ NK cells but not to CD19+ B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.     

Interaction of soluble CD163 with activated T lymphocytes involves its association with non-muscle myosin heavy chain type A

CD163 is a monocyte/macrophage-specific scavenger receptor that undergoes ectodomain shedding upon an inflammatory stimulus. Soluble CD163 (sCD163) actively inhibits lymphocyte proliferation, but to date exactly how it interacts with these cells has remained elusive. We screened T lymphocytes and endothelial cells for proteins binding to sCD163. In both cell types a high affinity binding protein was detected. Partial sequencing of the protein revealed sequence identity to a non-muscle myosin heavy chain type A. Employing labelled sCD163 we found little specific binding of sCD163 to the extracellular domains of T lymphocytes and human umbilical vein endothelial cells (HUVEC). In activated T lymphocytes we demonstrated specific binding of sCD163 to intracellular structures as well as the presence of the native protein within the cell after co-incubation with purified sCD163. Furthermore, we developed a novel ELISA for highly specific detection of sCD163-myosin complexes. These complexes were present in activated T lymphocytes after incubation with shed sCD163. Co-localization of sCD163 and cellular myosin in T lymphocytes was further confirmed by fluorescence microscopy. Our results suggest that sCD163 associates with cellular myosin, thereby possibly modulating the cells' response to an inflammatory stimulus.     

Pretransplant HRCT Characteristics Are Associated with Worse Outcome of Lung Transplantation for Cystic Fibrosis Patients

Objectives

Peri- and postoperative complications diminish the outcome of lung transplantation (LTx) in patients with cystic fibrosis (CF). We hypothesized that the degree of pathological findings on pre-LTx high resolution computed tomography (HRCT) is associated with higher morbidity and mortality in CF.

Methods

All our CF patients undergoing LTx between 2001 and 2011 were included. HRCT examinations were evaluated according to a scoring system for pulmonary disease in CF patients, the Severe Advanced Lung Disease (SALD) score and for pleural involvement.

Results

Fifty-three patients were included. Dominant infectious/inflammatory disease according to the SALD score was observed in 10 patients (19%). Five (50%) of those patients died within one week after LTx, compared to 2 (5%) patients without dominant infectious/inflammatory disease (p<0.001). This difference in survival percentage remained also significant in multivariate analysis. Patients with infectious/inflammatory disease received more packed red blood cells; 26 versus 8 in the first week (p<0.001). Pleural thickening was associated with higher requirement (10 units) for blood transfusion during LTx, compared to patients with normal pleura (4 units).

Conclusions

The analysis of HRCT in CF patients according to the SALD score showed that dominant infectious/inflammatory disease is associated with a higher mortality after LTx. If confirmed in other studies, HRCT might aid estimation of surgical risk in some adult CF patients.