Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Molecular characterization of the variable regions of a mouse polyreactive IgG2b antibody with rheumatoid factor activity

The complete nucleotide sequences of heavy and light chains of a mouse polyreactive IgG2b antibody were determined. This antibody, obtained after primary immunization of BALB/c mice with human lymphoblastoid cells, possess anti-HLA-DR and anti-rheumatoid factor activities and reacts with various self and nonself antigens. The VL and VH segments were found to belong to the VK8 and VH7183 families, respectively. The VH segment shared a high percentage of sequence similarity (95%) with previously described germline genes. The VK segment had 98.9% of sequence similarity with a consensus sequence VK8 of antibodies with anti-phosphorylcholine activity. Furthermore, the framework regions 2 and 3 of the VL segment were very similar to the framework regions 2 and 3 of other antibodies known to possess rheumatoid factor activity. We postulate that during immunization, the presence of HLA-DR antigens selects precursors having configurations similar to that of the germline, and induces some somatic mutations that do not significantly affect antibody polyreactivity.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X53400 and X59816 for VH and VL respectively.     

Modulation by zinc chloride of concanavalin A binding to rat thymocytes and early inhibition of lectin-induced blastogenesis.

Zinc chloride was shown to modulate the number and mobility of concanavalin A receptors of rat thymocytes at 4 °C. Zinc was able to induce the exposition at the cell surface of the same number of receptors that can be exposed by high doses of concanavalin A. It only weakly restricted their mobility. Moreover, zinc chloride was shown to inhibit rat thymocyte stimulation by succinyl-concanavalin A. This inhibition occurred most effectively if zinc was present during the first few hours after mitogen addition to cell cultures, as assessed by [³H]uridine incorporation in RNA and [³H]thymidine incorporation in DNA. The time at which DNA synthesis took place was not modified by zinc. Blast transformation in the presence of zinc was characterized by fewer blast cells which otherwise appeared identical to blast cells in control cultures. The possible action on membrane-cytoskeleton interactions is discussed.     

Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens

Spleen cells from nonimmunized BALB/c mice were fused with two nonsecreting myeloma lines. The hybrids were selected in HAT medium and screened for Ig production and for antibody activity against actin, tubulin, myosin, thyroglobulin, myoglobin, spectrin, dsDNA, fetuin, and transferrin. Among 161 hybrids secreting Ig, three were found to react with DNA, one with thyroglobulin, and one mainly with myosin. Two of these hybrids could be propagated and further characterized. On the basis of inhibition experiments, one was found to be directed against dsDNA; the other was directed mainly against myosin but at the same time reacted significantly with actin, tubulin, spectrin, and dsDNA. Reactivity with myosin seemed to be concentrated in the light meromyosin subfragment, known to be rich in alpha-helical structure. These results indicate: 1) There are reactive B cell clones directed against self antigens. 2) The antibody specificities found for these antibodies are very similar to those found for natural antibodies in normal human serum and for human monoclonal Ig. 3) The widespread reactivity found for the clone mainly reacting with myosin raises the possibility that the determinant recognized by this antibody is a conformational structure that possibly is associated with alpha-helical structures.     

PLASMA MEMBRANE AND INTERNALIZED IMMUNOGLOBULINS OF LYMPH NODE CELLS STUDIED WITH CONJUGATES OF ANTIBODY OR ITS FAB FRAGMENTS WITH HORSERADISH PEROXIDASE

Normal rat and mouse lymphoid cells were incubated at 0°–4°C for 1 h with purified rabbit or sheep antirat (mouse) immunoglobulin (Ig)-horseradish peroxidase (PO) conjugates or with Fab fragments of antibody coupled with peroxidase. Cells were subsequently washed and incubated in fresh medium, without labeled antibody or Fab fragments for 5–30 min at 20° or 37°C. With the use of the diaminobenzidine (DAB) method, distribution of peroxidase was studied in the light and electron microscopes. Fab fragments of antirat Ig antibody were iodinated with ¹²⁵I and subsequently coupled with horseradish PO. Plasma membrane and internalized immunoglobulins were detected by electron microscope autoradiography and peroxidase cytochemistry. Single- (Fab-PO), and double- ([¹²⁵I]Fab-PO) labeled lymphoid cells showed identical patterns of surface or internal distribution of immunoglobulins. In the electron microscope, Fab-PO conjugates at 0°–4°C resulted in a diffuse specific staining of the plasmalemma of lymphocytes and plasma cells. Most of the small dark lymphocytes (T cells?) did not show plasma membrane Ig. Macrophages did not show plasmalemma staining, but displayed nonspecific cytoplasmic staining after incubation at 20° or 37°C with antibody or Fab-PO conjugates. Lymphocytes and plasma cells, after incubation with antibody-PO conjugates at 0°–4°C, had patchy deposits of oxidized DAB on their plasma membranes. Macrophages, similarly treated, had no plasmalemmal staining. Patch and cap formation on the plasma membrane of lymphocytes and plasma cells was seen regularly after antibody-PO incubation at 37°C. Internalization patterns were different in lymphocytes and plasma cells. In lymphocytes, peroxidase staining was observed in small round or oval vesicles clustered at one pole of the cell (30 min at 37°C). In plasma cells, peroxidase staining was seen in clusters of tubules resembling the Golgi apparatus. Internalization of plasma membrane IgG was less pronounced after antibody-PO labeling as compared to Fab-PO labeling.