Biomedical subjects
Publications and source records attributed to A Hamann.
Lymphocyte traffic seems to be an essential requirement for an adequate immune response both in lymphoid tissues and local inflammatory sites. In this review, Adrian Duijvestijn and Alf Hamann discuss how selective migration of lymphocytes is directed by lymphocyte-endothelial interactions and what mechanisms may control this.
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In a variety of lymphocyte interactions, lymphocyte function-associated antigen-1 (LFA-1) plays an important role as an accessory mechanism mediating cell adhesion. We tested the possibility that LFA-1 could also be involved in the specific binding of lymphocytes to high endothelial venules (HEV) during homing. Antibodies against LFA-1 but not against various other cell surface molecules (except the putative gp90 homing receptor defined by the MEL-14 antibody) were found to inhibit in vitro adherence of lymphocytes to HEV in frozen sections of lymph nodes. Binding of T cell lines to HEV was also inhibited by anti-LFA-1 antibody. Using sublines selected for differential expression of the MEL-14 antigen, MEL-14 high cells (which bind well to HEV) were less susceptible to inhibition by anti-LFA-1 than poor binders with low levels of the homing receptor, supporting the model of LFA-1 being an accessory mechanism strengthening weak interactions between cells. Parallel results were found in vivo where anti-LFA-1 antibodies reduced the migration of normal lymphocytes into lymph nodes and Peyer's patches by 40 to 60%. Localization in the lung, especially of activated lymphocytes, was also impaired, although to a lesser extent. These findings suggest that LFA-1 plays an accessory role in cellular interactions relevant for lymphocyte migration.
Upon activation, lymphocytes display profound alterations in their in vivo migration behavior. In an attempt to understand some of the cellular mechanisms responsible for this altered behavior, in vitro stimulated lymphocytes have been analyzed for their expression of a putative homing receptor (HOR) (defined by mAb MEL-14) and for their ability to bind to specialized lymphoid organ high endothelial venules (HEV) in vitro. The results indicate that signals related to lymphocyte activation induce complex alterations in HOR expression and organ-specificity of HEV-binding: 1) submitogenic stimuli induce an increase in MEL-14 antigen expression. This applies to almost all lymphocytes in autologous cultures, for the fraction of cells in periodate, LPS- or Con A-treated cultures not fully activated and for cultures stimulated with suboptimal doses of Con A. 2) Full blast transformation is associated with a decrease or complete loss of MEL-14 antigen expression on the majority of blasts in all activating systems used, but a subset of up to 30 to 40% of fully activated cells may nonetheless express very high levels of the MEL-14 antigen. 3) Functional assays reveal that Con A and periodate stimulation lead to a selective, nearly complete suppression of the lymphocytes binding to HEV of Peyer's patches, even under conditions where overall binding to peripheral node HEV is increased. This indicates a differential regulation of the two respective receptors, with the mucosa system-specific HOR being more prone to down-regulation during in vitro activation by these mitogens.
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The experiments show that homing receptors are regulated in a complex fashion during initial cellular activation: Signals leading to blast formation induced either: --a decrease of homing receptor expression in the majority of blasts; --an increase of the Mel-14 expression in 20-40% of the blasts, and --a selective down-regulation in the capacity to bind to Peyer's patch HEV even under conditions, where binding to peripheral HEV is high. Submitogenic stimuli in partially activated cultures induce a rise in Mel-14 antigen expression and binding to peripheral node HEV, whereas Peyer's patch binding is unchanged or lowered. Thus, a selective and differential regulation of organ-specific homing receptors takes place under distinct activation conditions. The mucosal system-related receptor is more easily down-regulated upon activation. The in vivo homing experiments indicate that mitogen activation induces one dominant migratory phenotype. Alterations in homing receptor expression seem to be associated with changes in further cellular functions leading to reduced entry into lymphatic tissues and increased localization of these cells in lung or liver. The mechanisms regulating the differential expression of organ-specific homing receptors and additional homing-relevant properties of the cells are still unknown.
The position on the Escherichia coli chromosome of trkA, a gene coding for a membrane protein involved in K+ transport by the constitutive uptake system Trk, was determined. We observed that the gene is transcribed in a clockwise direction and that it is located at 72.4 min on the chromosome in a 1.75-kilobase NruI-EcoRV DNA fragment 1.0 kilobase upstream of rplQ. We localized an additional gene encoding a 17,000-molecular-weight protein of unknown function between the trkA and rplQ genes. A plasmid, pDB3, was constructed in which the transcription of the trkA gene was put under the control of the lambda pL promoter. pDB3-containing cells of a strain, which contained the temperature-sensitive lambda repressor cI857 in the chromosome, overproduced the 53,000-molecular-weight TrkA protein at the nonpermissive temperature to such an extent that TrkA became the major cell protein. From cell fractionation studies, we conclude that the overproduced TrkA protein forms aggregates.
When lymphocytes are activated in vitro, discrete cell-cell contacts are initiated which result in cluster formation. This contact interaction is found in syngeneic or allogeneic mixed leukocyte reactions as well as in mitogen-stimulated cultures (concanavalin A, periodate, lipopolysaccharide). T cells as well as B cells display the binding phenomenon. This activation-dependent lymphocyte-lymphocyte adhesion involves LFA-1, since monoclonal antibodies (including Fab fragments) against this molecule inhibit adhesion between clustering lymphocytes in a dose-dependent manner, whereas antibodies directed to several other cell surface antigens are inactive. Since a wide variety of functional interactions are inhibited by antibodies to LFA-1, it may be concluded that LFA-1-mediated cell contact is a discrete and essential step between a recognition event and the generation of functional activities by lymphocytes in general.
This study presents the partial molecular characterization of the alloantigenic rat T-cell marker RT-6. In contrast to most lymphocyte surface membrane proteins, RT-6 proved to be resistant to short-term (15 min) solubilization by TX-100 at 4 degrees, but could be efficiently solubilized by long-term (16 hr) exposure at 4 degrees or short-term (15 min) exposure at 37 degrees. SDS-PAGE analyses of RT-6.2 immunoprecipitates under non-reducing conditions revealed two bands with apparent molecular weights of 21,000 and 24,000 (25,000 and 28,000 under reducing conditions). Radiolabelled sugars failed to be incorporated metabolically into RT-6.2, neither endoglycosidase-F and 0-glycanase digestion nor NaOH treatment caused any detectable decrease in the molecular weight of RT-6.2, and RT-6.2 failed to bind to any of a series of agarose-coupled lectins with a wide of sugar-binding specificities. These observations suggest that RT-6.2 may lack carbohydrate moieties.
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Cell contact between lymphocytes can be observed in the form of clustering in autologous cultures of rat or mouse lymph node cells. Mutual binding takes place in the absence of adherent cells and is displayed by B cells as well as by T cells, with the exception of immature (Lyt 1,2+) T cells. Contact formation is related to activation of the lymphocytes since thymidine-incorporating cells as well as plaque-forming cells are concentrated in the cluster cell fraction and the formation of clusters is greatly increased by periodate stimulation. The interaction is selective with respect to cell type (cells of other tissue origin are not bound) and differentiation (only activated lymphocytes and some of several lymphoid cell lines are able to interact). The reaction is not genetically restricted, but takes place even between different (but related) species. Neither antigen nor MHC structures are involved in contact formation. Protease treatment abolishes the ability to form clusters, but one part of the interacting receptor/acceptor structures is apparently trypsin resistant. The interaction is dependent on the presence of magnesium, whereas calcium ions have no supporting effect. Involvement of the cytoskeleton is shown by a partial inhibition of the cluster formation by cytochalasin B and azide. No indication for a lectin nature of the binding structures could be found by carbohydrate inhibition studies. The relation of this interaction mechanism to other models of physical interaction in the immune system as well as its possible function for signal exchange and local recruitment of activated cells is discussed.
A specific antiserum against bovine heart catalytic subunit was used for the determination of the catalytic subunit in an enzyme-linked immunosorbent assay. Under the conditions elaborated the assay has a lower detection limit for catalytic subunit of 0.25 pmol/ml. In crude bovine heart extracts the concentration of catalytic subunit was determined by this method to be 0.18 +/- 0.02 mumol/kg wet wt. The immunochemical comparison of various animal species and cells, including organisms like amoebae and yeast, shows the broad applicability of the assay and provides evidence that the catalytic subunit is a highly conserved molecule.
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The recently detected T axis differentiation antigen, which was provisionally designated as T-lymphocyte-macrophage-associated antigen (TLMA) could now be identified as the neutral glycosphingolipid GgOse4Cer (asialo-GM1). TLMA is not only expressed on lymphocytes on the T lineage and on macrophages of the rat, but also on eosinophilic cells. On erythrocytes, the determinants are only detectable after neuraminidase treatment. Within the cell surface of thymic lymphocytes, the antigen determinant is partly masked by sialic acid residues. Presently, it cannot be decided whether the masking effect is brought about by sialic acid residues of adjacent glycolipid molecules or whether a nonreactive sialylated precursor molecule exists.
Antisera were raised in rabbits to an antigenic structure present in fetal pancreas tissue, pancreatic tumor tissue, and pancreatic juices and in sera obtained from pancreatic cancer patients. The first chemical data indicate that this pancreatic oncofetal antigen is distinct from CEA, NCA, and NCA2 and is not glycolipid in nature. Immunoelectrophoretic analyses demonstrate that pancreatic oncofetal antigen is a protein or a glycoprotein, which displays microheterogeneity. The apparent molecular weight of the basic unit of pancreatic oncofetal antigen is estimated to about 40 K. So far 234 pancreatic juices have been investigated for pancreatic oncofetal antigen. Pancreatic oncofetal antigen was detected within the pancreatic juices from 59 of 74 (80%) pancreatic carcinoma patients but could be traced in only 16%-26% of samples obtained from patients with other diseases.
An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.
1. The human thymus-brain antigen, which corresponds to the murine (mouse or rat) Thy-1 antigen complex, was isolated from brain after solubilization in deoxycholate by gel-permeation chromatography, wheat-germ-lectin affinity chromatography and ion-exchange chromatography. 2. The isolated antigen is a glycoprotein displaying an apparent molecular weight of 26 000-29 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. No antigen activity was found with the lipid fraction from human brain. 4. The protein has a tendency for spontaneous self-association (dimerization), leading to aggregates resistant to dissociating and reducing agents on prolonged storage. 5. The antigen is microheterogeneous with respect to size, charge (approximate isoelectric points of the monomer 7.7, 7.0 and 6.5) and to lectin-binding affinity. 6. The antigen can be reconstituted to protein-lipid vesicles. The antigen activity of solubilized antigen is strongly increased by reconstitution and that of membranes decreased by solubilization with detergent.