Surface immunoglobulin on rabbit lymphoid cells. II. Ultrastructural distribution of b4 allotypic determinants and morphology of spleen lymphocytes

Surface immunoglobulin allotypic determinants on rabbit spleen lymphoid cells are ultrastructurally localized by labeling with antiallotype antisera and soluble complexes of ferritin and rabbit antiferritin of a given allotype. At 0 °C surface Ig is visualized in patches on the membrane of 54% of the spleen lymphocytes examined. Four morphologically distinct categories of spleen lymphocytes display different amounts of labeled surface Ig. Type I cells are essentially identical to peripheral blood lymphocytes and demonstrate rapid endocytosis of surface Ig at 37 °C. Type II cells greater amounts of surface Ig, demonstrate little endocytosis, and are consistent with lymphoblast cells. Type III cells have the greatest amount of surface Ig, reveal some endocytosis, and are morphologically consistent with proplasmacytes and plasmablast cells. Type IV cells are immature plasma cells and have very little detectable surface Ig. The percentage of each cell type making up the labeled population is Type I, 28%; Type II, 21%; Type III, 50%, and Type IV, 1%. Immunoferritin labeled Ig determinants may be modulated from the surface of these cells at 37 °C by endocytosis and/or by shedding after reaction with anti-Ig antisera.     

Surface immunoglobulin on rabbit lymphoid cells. III. Double expression and separate endocytosis of surface immunoglobulin allotypes on heterozygous lymphocytes demonstrated by immunoelectron microscopic labeling.

The expression of immunoglobulin b locus (k chain) allotypes on the surface of rabbit peripheral blood lymphocytes (PBL's) is examined using an indirect double immunoelectron microscopic labeling technique. Ferritin and whelk hemocyanin individually conjugated to allotypically specific IgG are used as ultrastructurally identifiable molecular markers. These indicators are coupled to lymphocyte surface immunoglobulin (Ig) allotypic determinants by an antiallotype antibody linkage. Human red blood cells, conjugated with IgG of a specific allotype and used as test cells, demonstrate the absolute specificity and high efficiency of the ultrastructural labeling technique. Specific labeling on rabbit PBL's shows that 65–75% of the cells are positive for surface Ig. Lymphocytes from homozygous donors (b4b4 or b6b6) are labeled specifically with only the appropriate allotypic labeling system. Thirty-three percent of the PBL's from heterozygous donors (b4b6) express both allotypes (allelic inclusion) on the cell surface; the remaining proportion of Ig-bearing cells have only one detectable allotype present (allelic exclusion). We conclude that approximately 50% of the Ig-bearing PBL's demonstrate allelic inclusion for the b locus allotypes. On allelically included heterozygous lymphocytes, both allotypic determinants can undergo specific endocytosis. Endocytosis of one allotype on heterozygous cells can be induced by stimulation with antiallotypic serum without affecting the surface appearance of the other allelic marker (separate endocytosis).     

Distribution of immunoglobulin determinants on the surface of Xenopus laevis splenic lymphocytes.

The distribution of surface immunoglobulin (Ig) determinants on Xenopus laevis splenic lymphocytes after combination with divalent rabbit anti-Ig coupled to ferritin was studied. The electron micrographs showed the presence of immune complexes in 67% of lymphocytes treated at 0 degrees C-4 degrees C. The complexes were located all around the membrane and uniformly distributed in a random fashion. The variation of ferritin grain counts on cell sections is such, that the existence of two major subclasses of Ig-positive cells may be suggested. Raising the temperature produced a rapid interiorization of the complexes in vesicles without any previous aggregation to form a "cap" having occurred.     

Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat.

Involvement of IgE antibody in macrophage cytotoxicity against Schistosoma mansoni schistosomula suggests a cytophilic interaction of this class of antibody with the membrane of macrophages. Peroxidase-labeled monoclonal IgE protein was used to investigate IgE-macrophage interaction in the rat. Benzidine-aggregated rat IgE bound to the surface of peritoneal macrophages under experimental conditions, preventing endocytosis of the labeled aggregates. Binding was inhibited by preincubation with unlabeled IgE (aggregated). When unaggregated IgE was used, labeling of the macrophage surface, even when endocytosis was inhibited, was also observed at 37 °C but not at 4 °C. This result indicated different binding characteristics than reported for cytophilic IgG. Radiolabeled monoclonal IgE (deaggregated by ultracentrifugation after labeling) bound to peritoneal rat macrophages at 37 °C with a maximum between 10 and 20 min and a progressive shedding thereafter, whereas no change in bound radioactivity was observed at 4 °C or after preincubation with unlabeled IgE. Radiolabeled bovine serum albumin as a control did not interact significantly with the macrophages at both temperatures in these experimental conditions. The use of ?-mono-specific rabbit anti-rat IgE allowed the identification of IgE on the surface of peritoneal macrophages from rats infected with S. mansoni.     

Xenopus laevis larval thymocytes do not express surface immunoglobulin

Xenopus laevis larval thymocytes and splenocytes were examined for the presence of Ig determinants by an indirect immunofluorescence technique, using rabbit antiserum to deglycosylated Xenopus immunoglobulins. Thymocytes had no detectable surface membrane Ig, while Ig determinants were identified on the surface of a large percentage of the lymphocytes from the spleen. The positive fluorescent staining that one obtains on the surface of thymocytes using antisera to intact Ig's is due to antibody molecules directed to the carbohydrate determinants of the Ig's which cross-react with thymocytes' surface carbohydrate determinants.     

Characteristics of the macrophage uptake of proteinase-α-macroglobulin complexes

Complexes formed between labelled proteolytic enzymes (trypsin, subtilopeptidase A) and the α-macroglobulins of plasma are rapidly and selectively taken up by rabbit alveolar macrophages. The uptake occurs oxver a narrow zone of pH. Kinetics of the uptake is affected by temperature; in particular, incubation of macrophages at 37° C before the addition of labeled complex reduces the capacity to take up complexes. EDTA prevents the association of labelled complexes with macrophages, and can dissociate previously bound label. The effect of EDTA is reversed by the addition of calcium or magnesium or both. Iodoacetamide does not prevent the uptale of complexes but causes them to remain available for dissociation from the cells by EDTA. Incubation of complexes with macrophages at 37° C with no iodoacetamide results in the appearance of trichloroacetic acid soluble products of the enzyme in the supernatant fluid. These observations indicate that the selective uptake of proteinase-α-macroglubin complexes with rabbit alveolar macrophages can be resolved into three phases: (1) membrane binding which depends upon divalent cations and is pH sensitive, (2) endocytosis inhibitable by iodoacetamide and (3) temperature-dependent hydrolysis of the contained labelled enzyme.     

Antigen presentation by hapten-specific B lymphocytes. IV. Comparative ability of B cells to present specific antigen and anti-immunoglobulin antibody

The ability of trinitrophenyl (TNP)-binding murine B lymphocytes to present native rabbit IgG (RGG), TNP-modified RGG, and rabbit anti-mouse Ig (RAMG) to an Ia-restricted, RGG-specific helper/inducer T cell clone was compared. By three independent assays (lymphokine secretion, T cell proliferation, and B cell differentiation), TNP-RGG was presented at 10(2)- to 10(3)-fold lower concentrations than RGG, and RAMG at 10(2)- to 10(3)-fold lower concentrations than TNP-RGG. The available data suggest that the efficiency of antigen presentation is dependent primarily on the avidity of binding of a ligand to B cell surface Ig and/or the extent of subsequent endocytosis (modulation). Despite the observed quantitative differences between anti-Ig (RAMG) and specific antigen (TNP-RGG), these results demonstrate that qualitatively both are essentially similar in their ability to mediate specific T-B interactions. Thus, anti-Ig antibodies are valid models for analyzing cognate interactions between antigen-specific B and helper T lymphocytes.     

Modulation and regrowth of allotype on normal rabbit peripheral blood lymphocytes: allelic inclusion of b4 and b6 in single cells.

Allelic inclusion at the b-locus by heterozygous peripheral blood rabbit lymphocytes was demonstrated by using the mixed antiglobulin techniques (6, 7, 19). Heterozygous cells (64, 6) were treated with monospecific antiallotype reagents at 4 °C and warmed at 37 °C. The removal of surface allotype determinants was studied and, both the b4-and b6-specificities co-modulated after sensitization with either anti-b4 or anti-b6. Experiments were undertaken in which cells stripped of one or both allotypes by antiallotype induced modulation were cultured overnight. Allotype was then regenerated. Double expression of allotype by cells before antiallotype treatment was recovered following overnight regrowth at levels equal to those seen before treatment. Such was not the case when b44 and b66 cells were cultured together. These results indicate that normal heterozygous peripheral blood lymphocytes may express both b-locus alleles and that these determinants are in some manner physically associated with one another in the cell membrane.     

Neoantigen of the complement membrane attack complex of cytotoxic human peripheral blood lymphocytes.

Neoantigenic determinants (neoAg) specific for the assembling membrane attack complex (MAC) of complement were detected by immunofluorescence microscopy on the surface of cytotoxic lymphocytes during the antibody-dependent cellular cytotoxicity (ADCC) reaction. This study employed antibody-sensitized chicken erythrocytes as target cells, human peripheral blood lymphocytes as effector cells, and RITC-conjugated rabbit F(ab')2-anti-neoAg. NeoAg was present on 60% of ADCC plaque-forming lymphocytes (PFL). Eight out of 182 neoAg-positive PFL were observed in direct contact with their target cells. In these cases MAC-specific neoAg was visualized at the zone of contact between the cells. Anti-neoAg Ig was found to inhibit ADCC plaque assays up to 62%; and 51Cr-release assays up to 79%. Stimulation of lymphocytes by PHA or mixed lymphocyte culture increased the expression of neoAg. In the case of PHA, increased neoAg expression was correlated with an increased incorporation of 14C-leucine into C5, C6, C7, and C8 antigens, which was detected by immunodiffusion and autoradiography.     

Redistribution of cell surface transferrin receptors prior to their concentration in coated pits as revealed by immunoferritin labels

Summary Immunocytochemistry has been used to study distribution of cell surface transferrin receptors in erythroid, leukemic (K562) cells. The cells were fixed and labelled with monoclonal (OKT-9) anti-transferrin receptor antibodies; the antibody-labelled receptors were then detected by either immunofluoresceinor immunoferritin-antimouse-antibody conjugates. Typically, the immunoferritin labels were distributed diffusely at the non-coated regions of the cell surface as well as concentrated in the clathrincoated pits. To examine further this pattern of distribution, cells were labelled at 0° C and then warmed to 37° C for zero to 30 min prior to fixation. The majority of the immunoferritin labels were initially dispersed in small groups at the non-coated regions of the cell surface (mean = 6 immunoferritin labels/cluster), but larger groups were common subsequent to incubation at 37° C (mean = 13 immunoferritin labels/cluster). However, the size of immunoferritin labels in the coated pits was unchanged (mean = 12 immunoferritin labels/pit). Immunoferritin labels were typical in coated and uncoated vesicles l min after warming to 37° C, but common in endosomes, multivesicular bodies and lysosomes by 30 min. It appears that single cell-surface receptors form large aggregates prior to their concentration in coated pits. Coated vesicles, uncoated vesicles, and endosomal vacuoles may together form the non-lysosomal compartment where the internalized receptors might be dissociated from the ligands (antibodies).     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

Role of lymphocyte surface determinants in lymph node homing

Thoracic duct lymphocytes briefly incubated in vitro with trypsin and then infused into syngeneic rats are unable to migrate into lymph nodes. Trypsin-treated lymphocytes incubated at 37 °C in the absence of enzyme for 12 hr recovered their lymph node homing properties. In vitro recovery did not occur if the cells were cultured at 17 °C. Evidence was obtained that trypsin cleaved sialyglycoproteins from the surface of lymphocytes and that these determinants reappeared after the cells were maintained at 37 °C for 24 hr.Puromycin added to cultures of normal lymphocytes for 3 hr before infusion markedly reduced the recovery of donor cells in lymph nodes. The results suggest that surface determinants of recirculating lymphocytes essential for homing into lymph nodes may be rapidly turned over.     

Studies on the recognition of xenogeneic cells by nonimmune macrophages. I. Destruction of chicken fibroblasts in vitro by murine macrophages.

Human cord blood lymphocytes were compared with adult lymphocytes with regard to proportions of cells with surface markers for surface immunoglobulin (Ig), receptors for C′3 and the Fc-portion of IgG, as well as two types of erythrocyte rosettes (rapid and late E-rosettes). A significant decrease (P < 0.02 ? 0.05) in both early and late E-rosettes was noted when cord cells were compared to adult lymphocytes. After 20 hr of incubation at 37 °C, proportions of cells bearing Fc receptors in cord blood samples showed striking increments (P < 0.001) when compared with adult lymphocytes. T cell enrichment studies and sequential depletion of cells bearing Fc receptors as well as E-rosette forming cells indicated that the precursors of cells generating Fc receptors in vitro did not arise from cells with Fc receptors or T cell markers.     

Emergence of a surface immunoglobulin recycling process during B lymphocyte differentiation

Surface immunoglobulin (Ig)-mediated endocytosis has been investigated in rat B lymphocytes and plasma cells, using horseradish peroxidase (HRP)-labeled sheep anti-rat Ig Fab' fragment of antibody and HRP as monomeric ligands, respectively. Quantitative estimates of HRP activity associated either with plasma membrane or with endomembrane compartments were made in several experimental conditions. Binding of HRP-conjugate on B lymphocytes was followed by its endocytosis in combination with surface Ig, as shown by the progressive disappearance of plasma membrane-associated HRP activity. Between 1 and 6 h at 37 degrees C in presence of conjugate the total amount of cell-associated activity was constant. These results indicate that during this time no reappearance of surface Ig occurred by neosynthesis, by the expression of an intracellular pool or by the recycling in a free form of the previously internalized molecules. On the contrary, at saturating doses, internalization of HRP by anti-HRP plasma cells increased linearly with time at 37 degrees C in presence of antigen, when, during the same time, the plasma membrane HRP-binding capacity remained constant. Cycloheximide did not affect continuous HRP uptake. The existence of a large intracellular pool of receptors has been ruled out by experiments of removal of binding sites with pronase. In addition, monensin caused a progressive decrease in the number of surface receptors on plasma cells but not on B lymphocytes. Our data then indicate that, unlike B lymphocytes, plasma cells were able to recycle their surface Ig.     

Migratory patterns of B lymphocytes. IV. Effect of anti-Ig on follicular localization of radioactively labeled murine spleen and bone marrow cells.

A study was made of the localization of nylon-wool-adherent (AD) and nonadherent (NA) murine spleen cells in lymphoid tissue of irradiated syngeneic recipients. Cells were labeled in vitro with [³H]uridine or ⁵¹Cr and injected intravenously. Localization in recipient tissues was expressed as percent of injected radioactivity. NA and AD [³H]uridine labeled cells gave spleen to lymph node (S:LN) ratios of 1.0 and 2.7, respectively. After treatment of AD cells with rabbit anti-mouse Fab + C at 37 °C, localization in S decreased markedly.NA cells primarily localized in LN paracortex and splenic periarteriolar sheaths. Untreated and NRS-treated AD cells localized in lymphoid follicles, whereas anti-Fab-treated AD cells did not. When ⁵¹Cr-labeled AD cells were treated with anti-Fab at 4 °C without C, there was a transient decrease in splenic localization at 24 hr followed by a recovery to the normal pattern at 48 hr after transfer. [³H]uridine-labeled bone marrow (BM) cells showed less localization in lymphoid tissue than did S cells. Some BM cells were seen in LN follicles, particularly at 48 hr after transfer, but this localization was not affected by prior treatment with anti-Fab + C. The possible role of surface Ig in the determination of follicular localization of B lymphocytes is discussed.     

Further characterization of a novel lectin derived from silkworm faeces; specific binding to immunoglobulins,and activation of immunocytes

In previous studies a novel lectin-like glycoprotein was isolated from silkworm faeces and shown to recognize sugar chains, especially mannose on the cell surface as an epitope, and to cause aggregation of various types of cells in suspension. However, this substance caused detachment and aggregation of only some types of plated cells, such as QM-RSV cells, which are quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV) at 35.5 °C, a permissive temperature for RSV. As described here, during studies on the mechanism of cell detachment and aggregation of QM-RSV cells by this new lectin, some novel biological activities of the lectin were recognized. This lectin was found to bind to immunoglobulins (Ig) specifically at specific amino acid sequences, not via recognition of their molecular conformation. It also recognized the neural cell adhesion molecule (NCAM), which is one of the members of the Ig-superfamily that have Ig-like domains. Furthermore, it had a strong mitogenic effect on lymphocytes, and also caused about 3-fold of phagocytosis by macrophages within 24 hr after its addition.     

Redistribution of Surface Antigens– a General Property of Animal Cells?

MEMBRANE antigens of animal cells are reported to be localized either in discontinuous discrete areas (H-2^1,2, HL-A³, organ⁴ and immunoglobulin (Ig)^5–9 or continuously (species⁴ and blood group^9,10). These results have been obtained by immunofluorescence (IFL) and electron microscopy performed by double layer principles except in the case of Ig where direct IFL technique has been used. The influence on the localization pattern of marker molecules by variables such as Ig concentration of the reagents used, effects of interactions between Ig molecules in the double layer and temperature dependence of the reaction steps have not been investigated. Redistribution of lymphocyte surface Ig molecules induced by anti-Ig antibody has been described and this was observed to be temperature dependent occurring at 20° C but not at 0° C^11,12. Here I discuss the distribution and localization of different surface antigens (blood group substance A and B, species, H-2 and organ) on several cell types (monkey kidney, human lymphoid cells, HeLa, human thyroid cells, RK13 and L-cells of C3H/K) as recorded by direct and indirect IFL technique. I show that the temperature and Ig concentration used exert an influence on the distribution of these antigens. The cells used in IFL tests were cultured and harvested as described before¹⁰. IFL staining was performed according to Möller1³. The stained cells were examined with a Leitz ‘Orthoplan’ ultraviolet microscope. Direct IFL technique was applied to two test systems. A FITC-conjugated rabbit antiserum to blood group substance A was used on monkey (Macaca fascicularis) kidney cells known to be A-positive¹⁰ and a FITC-conjugated horse anti-human thymocyte globulin were tested on a human lymphoid cell line Robinson (obtained from Dr Moore, Buffalo). In both test systems the staining pattern was completely confluent at all temperatures used: 4° C, 20° C and 37° C. The staining pattern was not related to antibody concentration.     

Mycoplasma contamination of membrane associated measles antigens. Inability to demonstrate in vitro lymphocyte responsiveness to measles.

A marked suppression of protein synthesis including IgA was demonstrated in BALB/c mouse thymocytes incubated in short-term tissue culture in the presence of anti-mouse α chain antibodies. In C57BL mouse thymocytes a suppression of protein synthesis including IgM was obtained by incubating the cells in tissue culture containing anti-mouse μ chain antibodies. The association of anti-α chain antibodies or anti-κ chain antibodies, at 4 °, to the Ig subunits on the surface of BALB/c mouse thymocytes was demonstrated following radioiodination of the antibody treated cells. The anti-mouse Ig antibodies attached to the surface α chains or κ chains at 4 ° were radioiodinated on the cell surface, rendering the corresponding surface mouse Ig subunits inaccessible to radioiodination by lactoperoxidase. The α chains or κ chains complexed with their specific antibodies were independently removed from the cell surface upon heating to 37 °. However, it was found that each of the antibodies attached first to its Ig subunit on the cell surface interferes with the subsequent binding of antibodies to the second subunit. The antibody-surface Ig complexes, although cleared from the cell membrane at 37 °, were not released into the culture medium, leading to the conclusion that these complexes were endocytosed.     

Interaction and release of soulble immune complexes from mouse B luymphocytes

Soluble immune complexes (¹²⁵I BSA-anti-BSA-C) bind to B lymphocytes and accumulate at one pole of the cells (“caps”). The complexes remain on the membrane after incubation of the cells at 37 °C in tissue culture medium for several hours. The ¹²⁵I BSA can be quantitatively removed from the cell surface by incubation with excess BSA but not with excess antibody to BSA or preformed BSA-anti-BSA-C complexes. The release of ¹²⁵I BSA is probably due to the removal of the complexes from the cell membrane and not to an exchange between unlabeled BSA in the medium and the labeled BSA present in the membrane-bound complexes. Release of ¹²⁵I BSA by excess BSA is temperature dependent. The membrane-bound complexes can also be removed by incubating the cells with papain fragments of rabbit antibody to mouse Ig (anti-γ₁, γ₂, and k Ig chains). However, after exposure to divalent [F(ab′)² or 7S Ig] rabbit antibodies to mouse Ig, the complexes remain associated with the cells. In addition, after such treatment the complexes cannot be removed by excess BSA or by Fab anti-Ig.     

Functional properties of T and B cells isolated by affinity chromatography from rat thoracic duct lymph.

Rat thoracic duct lymphocytes (TDL) were separated into two fractions by passing the cells through a column of rabbit anti-rat F (ab′)₂ antibody coupled to Sephadex G-200. Cells with readily detectable surface immunoglobulin (Ig) were retained on the gel, whereas those without surface Ig were recovered in the effluent. Adherent cells were retrieved by eluting the column with rat Ig. Both dividing and nondividing lymphocytes were separated by this procedure. The adherent and non-adherent fractions contained functionally active lymphocytes as judged by a thymidine incorporation technique and the immunological performance of the cells after transfer to normal recipients. Antibody forming cells and B memory cells were concentrated in the adherent fraction. The non-adherent fraction contained antigen-sensitive T cells which initiate graft versus host reaction and specifically sensitized lymphocytes of the kind which transfer resistance to L. monocytogenes.