Sten Hammarström

The Interaction of Nonmitogenic and Mitogenic Lectins with T Lymphocytes: Association of Cellular Receptor Sites

The relationship between the surface receptors on neuraminidase‐treated human blood lymphocytes for the mitogenic lectins Phaseolus vulgaris leukoagglutnin (La), concanavalin A (Con A), and soy bean agglutinin (SBA) and the nonmitogenic lectin Helix Pomatia A hemagglutinin (HP) was investigated. Two different techniques, co‐capping with different fluorochrome‐labeled lectins and cell binding‐inhibition experiments with 125 I‐labled lectins, were used. The results demonstrated that the nonmitogenic lectin HP and the mitogenic lectins SBA, La, and Con A bind either to the same macromolecule(s) or to different but physically linked macromolecules on the surface of human T lymphocytes. In contrast, only part of β2‐microglobulin (β2‐m), or β2‐m‐bearing complexes, appear to be physically linked to the lectin receptor complex(es). On the lectin‐binding substance(s) at least two saccharide structures were recognized, one of which binds both HP and SBA and another which hinds SBA and La (and probably also Con A) but not HP.

ANTIGEN FROM COLON OF GERMFREE RATS AND ANTIBODIES IN HUMAN ULCERATIVE COLITIS

Evidence has been brought forward previously tha t sera from patients with ulcerative colitis contain circulating antibodies reacting with extracts from colon of newborn children who died without feeding during the first day of their life.’ Moreover, it has also been shown that white blood cells, probably lymphocytes, from the peripheral blood of such patients, exerted an immunologically specific cytotoxic effect in uitro on isolated fetal colon ’ Although these findings suggested that immune mechanisms might be responsible for tissue damage in ulcerative colitis, they gave no clue as to the origin of the sensitization. In view of the many alternative ways in which a sensitization against colon tissue can be assumed to arise,5 it was felt that a better knowledge of the nature of the antigens and antibodies involved was of primary importance as a basis for further studies. Because of its inherent contamination with antigens from the intestinal flora, colon tissue constitutes an extremely unfavorable object for immunological studies. Although this difficulty can be circumvented by the use of sterile newborn colon, such specimens cannot always be obtained in a state sufficiently fresh for antigen preparation and the amounts of active extracts will mostly be insufficient for systematic studies. This paper presents the results of an immunological study performed with extracts of the large intestine from germfree rats and sera from ulcerative colitis patients or various controls. I t was found tha t these extracts indeed contained antigen reacting with the patients’ sera. Therefore, further studies were conducted in order to establish the possible relationship between rat and human colon antigen. At the same time, the reactivity of extracts of germfree rat origin was compared with those obtained from conventional (nonsterile) intestines. Furthermore, the immunological relationship between

Autoantibodies to Colon in Rats and Human Ulcerative Colitis Cross Reactivity with Escherichia coli O:4 Antigen

Summary Autoantibodies to germ free rat colon were produced in rats by repeated intraperitoneal injections with newborn rabbit colon in Freunds complete adjuvant. Antibodies were assayed by means of indirect hemagglutination of sheep erythrocytes sensitized with a heat stable phenol-water extract of germ free rat colon or by fluorescent antibody staining of rat colon sections. The response of rat colon injected rats was less intense. A few of the rats injected with adjuvant alone also developed an anti-colon titer. Hemagglutination inhibition experiments demonstrated that the antibodies were specific for gastrointestinal antigen and represented autoantibodies in a true sense. They were found to be specific for at least three different colon determinants: one unique for germ free rat colon, one common for rat and rabbit colon, and one common for both colons and a polysaccharide from Eschericria coli O:14, grown on synthetic medium. No or only very weak cross reactions appeared with similar antigens extracted from a number of other E. coli strains. Old Tuberculin and extract of Staphylococcus aureus were inactive. The pattern of reactivity of the rat antibodies was similar to that of patients with ulcerative colitis. Stimulation by cross reacting bacterial antigen may be an important contributing factor for autoantibody formation in this disease.

Structure, specificity, binding properties, and some biological activities of a blood group A-reactive hemagglutinin from the snail Helix pomatia.

During recent years a considerable body of data has been accumulated on the specificity, molecular structure, and various biological properties of agglutinins (lectins) isolated from plants (for references, see Sharon and Lis l). In contrast, relatively little is known about agglutinins from invertebrates. Only a few have been purified and their chemical and physicochemical properties investigated, notably Limulus polyphemus hemagglutinin,2v Asterias forbesi aggl~t in in ,~ an agglutinin from the Murray mussel (Velesunio ambiquus) ,4 and Helix pomatia anti-A hemagglutinin.5, The specificity of the binding site has been investigated only for Helix pomatia anti-A hemaggl~tinin.~. It is the purpose of this review to describe the isolation procedure, the subunit structure, and the specificity and binding properties of Helix pomatia anti-A hemagglutinin. In addition, some recent data will be given on the biological effects of the agglutinin on human lymphocytes: e.g., its ability to stimulate lymphocytes to blast transformation. *

Purification and properties of biliary glycoprotein I (BGP I). Immunochemical relationship to carcinoembryonic antigen.

Abstract Normal human bile contained a biliary glycoprotein (BGPI), which was immunologically crossreactive with carcinoembryonic antigen (CEA). BGP I was purified by perchloric acid precipitation (1 M ), ion exchange chromotography, concanavalin A-Sepharose affinity chromatography and gelfiltration. Purified BGP I had a mol. wt of ~ 90,000 (SDS-PAGE and gelfiltration in 6 M guanidine-HCl) and contained about 40% carbohydrate. The same sugars and amino acids in similar molar proportions as in CEA were detected. The immunological cross-reactivity between BGP I, CEA and ‘non-specific crossreacting antigen’ (NCA) was established by immunodiffusion and immunoadsorbent experiments using antisera against the three antigens. BGP I differed from CEA and NCA in that it occured in normal human bile and that it contained unique antigenic determinants not present in CEA or NCA. Furthermore, most BGP I preparations lacked methionine in analogy with CEA but in contrast to NCA. A second CEA cross-reactive biliary glycoprotein of lower apparent mol. wt (~ 60,000, SDS-PAGE), termed BGP I low was also discovered in human bile. It was present in lower concentrations than BGP I. BGP I low was precipitated with BGP I antisera. It has not been established whether it is a degradation product of BGP I or still another member of the CEA glycoprotein family.

Receptors for Helix pomatia A Haemagglutinin (HP) on a Subpopulation of Human B Cells

Receptors for Helix pomatia A haemagglutinin (HP) have earlier been found on neuraminidase treated T‐lymphocytes in human peripheral blood. In contrast, the majority of the B‐lymphocytes, characterized by surface bound IgM and/or IgD (SIg) lack these receptors. Double marker experiments with fluorochrome labelled reagents have now shown that a minor fraction (3–24%) of the IgM/D bearing lymphocytes in normal human blood also have HP‐receptors. These HP+ B‐cells constitute approximately 1% of the HP+ lymphocytes in adult blood. Fractionation on HP‐Sepharose columns showed that the HP+ B‐cells are readily eluted with buffer containing 0.1 mg N‐acetyl‐D‐galactosamine. In contrast, the majority of the HP+‐Sig− cells require higher concentrations of the competing hapten for elution (1 mg d‐Ga1NAc/ml buffer). This indicates that the HP‐receptors on these B‐cells differ qualitatively or quantitatively from those on the majority of the T‐cells. Previous findings of HP‐receptors on the Sig+ leukaemic cells in the blood of patients with chronic lymphocytic leukaemia suggested that these structures are expressed on an immature variety of B‐cells. This assumption is favoured by the present finding that approximately 80% of the lymphocytes with surface bound IgM/D in cord blood also have HP‐receptors. Therefore, the HP‐receptor seems to fall in the category of differentiation markers and constitutes a useful tool for characterization and separation of human lymphocytes within both the T‐ and the B‐compartments.

Binding of Helix Pomatia A Hemagglutinin to Human Erythrocytes and their Cells. Influence of Multivalent Interaction on Affinity

The binding of 125I‐labelled intact (hexavalent) and partially reduced (divalent) Helix pomatia A hemagglutinin to human A.1, A.2, A.3, A1B and A2B erythrocytes, human lymphocytes, human lymphoblastoid and other tumor cell lines was investigated. The essential finding was that the association constants calculated for the interaction between hemagglutinin and A‐erythrocytes were many orders of magnitude higher than the intrinsic association constant for the interaction between the hemagglutinin and the blood group A determinant. The latter value was 5–1031/mole. The K‐values for intact hemagglutinin and A and AB erythrocytes were in the order of 10101/mole at 18–22 °C and pH 7.3. For partially reduced hemagglutinin the K‐values were in the order of 5–107 1/mole. Multivalent interaction would seem to be the essential factor responsible for the high K‐values in the cell binding experiments. Intact hemagglutinin reacted against A1 and A2 erythrocytes or against a human osteogenic sarcoma cell tine (2T) gave homogeneous binding curves in Scatchards plot. Human lymphocytes and most lymphoblastoid cell lines lacked hemagglutinin receptors.

Subunit structure of Helix pomatia A hemagglutinin.

Equilibrium centrifugation shows that the hemagglutinin has a mol. wt. of 79, 000 ± 4, 000. It contains approximately 18 moles of half cysteine, all as disulfide bonds. Unfolding agents alone (7 M guanidine.HCl) dissociate the hemagglutinin into a subunit of mol. wt. 26, 000–30, 000. Complete reduction (DTT in 7 M guanidine.HCl) gives a single component with a mol. wt. of approximately 13, 000. Partial reduction (DTT, 8 M urea or 7 M guanidine.HCl + protective haptens D‐GalNAc or D‐GNAC) cleaves 3 to 4 SS‐bonds giving a single component with a mol. wt. of 12, 500–16, 700. Tryptic digestion gives rise to 13–14 peptides, three of which contain cysteine. Partially reduced hemagglutinin contains an intact carbohydrate binding site. The data suggest that the hemagglutinin is made up of 6 identical or closely similar polypeptide chains, each containing 1 intrachain disulfide bond and 1 carbohydrate binding site. The subunits are arranged in pairs in which a single disulfide bond links the monomers together. Native hemagglutinin is formed by the interaction of non‐covalent forces between 3 dimers.

Quantitation of Salmonella O-Antibodies in Human Sera by Enzyme-Linked Immunosorbent Assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water extracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2, 12), B (4, 5, 12) and D (9, 12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid--or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater reproducibility.

Fractionation of Human Blood Lymphocytes on Helix pomatia A Haemagglutinin Coupled to Sepharose Beads

Abstract. Treatment of human blood lymphocytes with neuraminidase has previausly been shown to uncover receptors for the A haemagglutinin of the snail Helix pomatia (HP). Neuraminidase‐treated lymphocytes were now fractionated on columns charged with large Sepharose particles to which HP had been coupled covalently. HP‐receptor negative (HP‐) lymphocytes passed the columns while HP‐receptor positive (HP+) lymphocytes were retained. The latter cells were eluted by addition of the competitive hapten N‐acetyl‐D‐galactosamine (D‐GalNAc). The total yield of cells recovered after fractionation was 60‐80% Surface marker studies indicated that there was no selective loss of any of the major lymphocyte subpopulations. The fraction that passed the columns (fraction I) consisted of approximately 10% of all lymphocytes. It contained ˜ 1% HP+ cells and ˜3% of all lymphocytes forming rosettes with sheep erythrocytes (E+ cells) present before fractionation. 50‐55% of the lymphocytes in this fraction had surface‐bound immunoglobulin (SIg+ cells) and complement receptors (EAC+ cells). Of the SIg+ cells, ˜60% were true B cells while the remaining 40% had IgG adsorbed to their surface. The majority of the B cells were recovered in this fraction. The lymphocytes of this fraction responded poorly to T‐cell mitogen but had an enhanced K‐cell activity to chicken erythrocytes. Elution of the cells retained on the column with 0.1 mg/ml D‐GalNAc gave a fraction II, consisting of, ˜15% of all lymphocytes. This fraction had a mixed composition. The majority of the cells (˜45%) were recovered by subsequent elution with 1.0 mg/ml D‐GalNAc. This fraction III was strongly enriched with HP+ and E+ cells (T cells). About 10% of the HP+ cells in this fraction were SIg+. However, on the majority of these cells this surface‐bound immunoglobulin was probably externally adsorbed IgG. These HP+‐SIg+ cells were also EAC+ and had Fc receptors, as shown by rosette formation with IgG‐coated bovine erythrocytes. The lymphocytes of fraction III responded most strongly to T‐cell mitogen while their K‐cell activity was weak.