An explanation for the defect in secretion of IgM Mott cells and their predominant occurrence in the Ly-1 B cell compartment.

Mott cells are a variant form of plasma cell in which the immunoglobulin (Ig), rather than being secreted, accumulates in rough endoplasmic reticulum-derived vesicles called Russell bodies. We have examined the molecular cause of this defect and the in vivo origin of IgM Mott cells. Our examination of the Ig variable region gene sequences of two IgM Mott hybridomas derived from C.B-20 Ly-1 B cells showed all to be germ line. In a series of mix and match transfection experiments, the Mott phenotype was only reconstituted when the original Mott specificity was expressed as an IgM, suggesting that both the specificity and the isotype were critical to the formation of Russell bodies. Based on our finding that Russell body formation was dependent on the Ig isotype being IgM, we suggest that the Mott phenotype is apparent only after differentiation of B cells into plasma cells and that probably the major cause of the IgM Mott phenotype is low-affinity interaction of the Mott Ig with some as yet unknown intracellular component(s) being stabilized by the intrinsic high avidity of the pentameric secreted form of IgM. Consistent with this proposal was the finding that after in vitro lipopolysaccharide (LPS) stimulation of sorted Ly-1 B cells derived from C.B-20 mice, Mott cells represented up to 5% of the IgM plasma cells in the culture. LPS stimulation of conventional B cells also induced the appearance of IgM Mott cells, but at the much reduced level of 0.1%, suggesting that the major, if not the only, source of Mott cells in vivo is the Ly-1 B cell population. A possible causal relationship between the elevated frequency of Mott cells in the Ly-1 B cell-derived LPS blasts and the repertoire selection inherent in the development of these B cells is discussed.     

Mott cell tumor of the stomach with Helicobacter pylori infection

A plasma cell tumor of the stomach with unusual histology is reported. Macroscopically, the tumor formed two ulcers in the gastric body, and microscopic examination revealed proliferation of plasma cells producing immunoglobulin G kappa monotypic immunoglobulin, with metastatic infiltration in some perigastric lymph nodes. Most of these plasma cells had various-sized Russell bodies in the cytoplasm; hence the tumor may be called Mott cell tumor. The Russell bodies showed a strong affinity to concanavalin A by lectin immunohistochemistry, compared with those in reactive Mott cells. In addition, Helicobacter pylori (H. pylori) infection was proved by Gimenez stain and immunohistochemistry. The mixture of some centrocyte-like cells and presence of reactive lymph follicles with follicular colonization by tumor cells suggest that this lesion may be a variant of mucosa-associated lymphoid tissue lymphoma in association with H. pylori infection. The patient has shown no evidence of recurrence of the tumor after 11 years of follow up.     

Development of plasmacytoid cells with Russell bodies in autoimmune "viable motheaten" mice

Mice homozygous for the autosomal recessive mutation "viable motheaten" (mev) are severely immunodeficient, show polyclonal B-cell activation, and express multiple autoantibodies over a maximum life span of 25 weeks. Lymphoid tissues from these mice contain large numbers of atypical plasma cells in which discrete glycoprotein inclusions are found within the endoplasmic reticulum. Such plasma cells are termed "Mott cells," and the inclusions are called "Russell bodies." Dense accumulations of Mott cells are present in the marginal zones of the spleen and in the lymph nodes of mev/mev mice. Russell bodies in Mott cells from mev/mev mice contain immunoglobulin (Ig) as shown by immunofluorescence microscopy at the light-microscopic level and by indirect protein A-immunogold localization at the electron-microscopic level. Ultrastructural analyses reveal the presence of amorphous, lamellar, and crystalline Russell bodies. These Ig crystals have a periodicity of 150-190 A. Lymph node cell preparations which were enriched in Mott cells by velocity sedimentation failed to secrete Ig in a polyclonal reverse plaque assay. An obligate role of the thymus in Mott cell development is evidenced by the absence of Mott cells in neonatally thymectomized mev/mev mice and in mice doubly homozygous for the nude (nu) and mev mutations. These data suggest that Mott cells in mev/mev mice are thymic-dependent plasmacytoid cells resulting from chronic B-cell activation accompanied by impaired Ig secretion.     

Mott cells: a model to study immunoglobulin secretion

Mott cells are plasma cells defective in immunoglobulin (Ig) secretion. They display this defect by accumulating Ig in the rough endoplasmic reticulum, detectable by Ig+ intracellular inclusion. We have previously produced hybridoma cell lines (Alanen, A. et al., Eur. J. Immunol. 1985. 15: 235) in which this phenotype is preserved, and shown the inability of these cells to secrete the Ig. In order to study this defect further, we fused these hybridoma cells with a kappa-secreting hybridoma cell line, Sp1, and, using double selection with hypoxanthine, aminopterin, thymidine and ouabain, obtained hybrid cell lines expressing various combinations of the three Ig chains involved (Mott gamma 1, Mott kappa and Sp1 kappa chains). We studied the presence of Ig+ inclusions in these cells as well as Ig secretion by metabolic labeling and immunoprecipitation. All inclusion-positive clones expressed both Mott heavy and Mott light chains with or without the Sp1 light chain, whereas none of the inclusion-negative clones produced both Mott-derived Ig chains. In all of the clones, even those with inclusions, the Ig secretion was at least partially rescued by the fusion. This occurred also in an inclusion-positive clone which maintained the original Ig status of the Mott without Sp1 kappa chain, indicating a complementation by the cell fusion of some cellular factor involved in Ig secretion. Furthermore, we injected Xenopus oocytes with mRNA isolated from three different original Mott cell hybridomas and could show secretion of the Ig, which is not secreted from the original Mott cells, from the oocytes.     

Mott cells are plasma cells defective in immunoglobulin secretion

Plasma cells containing intracellular inclusions of immunoglobulin (Russell bodies) are known as Mott cells, and are found in large numbers in lymphoid organs in autoimmune mice. Hybridoma technique was used to produce cell lines of this phenotype by fusing spleen cells from a NZB mouse with a nonproducing hybridoma cell line (Sp2/0-Ag14), allowing us to carry out studies of this cell type at the biochemical level. Ultrastructurally the inclusions were distended cisternae of rough endoplasmic reticulum, suggesting a block in the secretory pathway of the cells. Biosynthetic labeling studies confirmed that these cell lines have either a complete or partial block of secretion of immunoglobulin, possibly due to an abnormal light chain.     

Expression of peroxiredoxins I and IV in multiple myeloma: association with immunoglobulin accumulation

B cell malignancies are classified according to the postulated differentiation stage of the originating cell. During differentiation, structural and molecular changes occur to support massive processing of immunoglobulin in the endoplasmic reticulum (ER) of plasma cells at the final stage. When overloaded, the ER generates unfolded proteins and hydrogen peroxide (H₂O₂), which may cause cell death. Peroxiredoxins (Prxs) I and IV belong to a family of proteins able to catalyze peroxide detoxification. Here, we investigated a potential association of these enzymes with immunoglobulin production in B cell neoplasms. Our results demonstrated that the expression of Prx IV was induced as cells became competent to synthesize immunoglobulin light chains, as observed by immunohistochemistry in tissue sections of B cell neoplasms and also by qPCR and Western blotting analyses in malignant B cell lines. Prx I was frequently highly expressed, indicating additional regulatory processes besides ER activity. Results obtained exclusively with myeloma cells have shown that expression of Prxs I and IV, both at mRNA and protein levels, was associated with light chain secretion quantified by ELISA. We suggest that Prxs I and IV may provide survival advantages for terminally differentiated neoplastic B cells by the elimination of H₂O₂ and, in the case of Prx IV, by the conversion of this toxic in a functional agent driving oxidative protein folding in the ER. In this sense, multiple myeloma and lymphomas demonstrated to synthesize immunoglobulin chains may benefit from strategic therapies targeting the adaptive pathway to ER stress, including inhibition of Prxs I and IV activity.     

Inhibition by Lysozyme of Growth of the Spherule Phase of Coccidioides immitis In Vitro

Development of mature endosporulating spherules from an endospore inoculum was markedly inhibited by human or hen egg-white (HEW) lysozyme at 5 μg/ml. Mature spherules formed in medium containing 5 μg per lysozyme per ml (3.3 × 10⁻⁷ M) were approximately 50% smaller than control spherules. In addition, lysozyme induced a large portion of the endospore inoculum to revert to the mycelial growth phase. Increasing lysozyme concentrations to 10 or 20 μg/ml prompted a nearly complete reversion of the inoculum to the mycelial phase. Mature endosporulating spherules removed from growth medium and resuspended in a solution of human or HEW lysozyme at 18 μg/ml in distilled water prompted leakage of four to five times as much of materials absorbing maximally at 260 nm into the supernatant as untreated control spherules during 90 min of incubation. This four- to fivefold increase in nucleotide loss was evident at 4, 25, and 37 C. The permeability of 1-day-old immature spherules and 8-day-old endospores was considerably altered by lysozyme treatment of cells suspended in distilled water. Large amounts of potassium and nucleotides were rapidly lost by each type of cell when treated with 20 μg of lysozyme per ml. After 270 min of exposure to lysozyme, 98% of the immature spherules and 25% of the endospores were nonviable. Lysozyme adsorption by formalin-killed spherules in the presence of varying concentrations of calcium ion and the rapid alteration of permeability seen after lysozyme treatment suggested that the cell membrane was damaged as a result of binding lysozyme.     

Significance of an in vitro phenomenon in which murine erythrocytes are lysed by autologous spleen cells and spherules of Coccidioides immitis.

Spleen cells from mice immunized with a variety of antigens and incubated in vitro with killed spherules of Coccidioides immitis lyse six to eight times more autologous murine erythrocytes than normal spleen cells and spherules. Cellular and biochemical events in this phenomenon were investigated to ascertain its significance. Kinetic studies suggested that hemolysis results from the activation of some immune cells by spherules. The capacity of spherules to activate these cells is rather unusual because, of the inert particles tested, only zymosan A and crude chitin demonstrated comparable activity. Furthermore, although the hemolytic phenomenon occurred in serum-free medium, more lysis was effected by immune cells and opsonized spherules or zymosan A than by immune cells and untreated fungal particles. Sheep, chicken, and human erythrocytes were not lysed in the hemolytic phenomenon; however, hemoglobin in chicken and sheep erythrocytes was oxidized. Both the murine erythrocyte lysis and oxidation of ovine hemoglobin correlated with the reduction of Nitro Blue Tetrazolium by immune cells adherent to spherules, and both phenomena appeared to be mediated by H2O2 released into the medium by activated cells. Spleen cells reactive with spherules could not be depleted by treatment with iron carbonyl, antiimmunoglobulin plus complement, or anti-brain-associated theta plus complement, but they were partially or completely depleted after rosette formation with erythrocytes coated with antibody or murine complement. Using light and electron microscopy, we noted that immune spleens contained more neutrophils than normal spleens, that these neutrophils reduced Nitro Blue Tetrazolium after stimulation with phorbol myristate acetate, and that they were the most prevalent cell type adherent to spherules after incubation in vitro.     

Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

Since the discovery of antibody‐producing B cells in chickens six decades ago, chickens have been a model for B‐cell development in gut‐associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJC_L knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL^?/?) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy‐chain‐only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4‐week‐old IgL^?/? chickens, and antigen‐specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy‐chain‐only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B‐cell development in a gut‐associated lymphoid tissue species.     

T Helper Cell Control of B Cell Development and Isotype Expression

The supernatant of the T helper cell clone 52.3 (52.3-SN) was shown to induce polyclonal activation of resting B cells. 52.3-SN acts on most small B cells and through the allogeneic barrier. This supernatant induces cell size increase, RNA and DNA synthesis, and appearance of interleukin-2 and transferrin receptor. These results are interpreted as indicating the existence of a B Cell Activating Factor (BCAF) acting on resting B cells in an MHC-unrestricted way.

TH cells can be obtained in an intermediate state of activation where they secrete lymphokines leading to B cell proliferation and not the biological activities leading to plasmocyte development. TH cell clones can induce sIgG^? and sIgA^? unprimed B cells to switch and express all classes and subclasses of immunoglobulin. The bulk of the response consists of IgM. Among the non IgM isotypes, IgG₁ and IgA predominate. The supernatants prepared from TH cells reproduce these effects.     

Affinity of immunoglobulin for heterologous tissue mast cells. A study with the fluorescent antibody method

A double layer immunofluorescence method was used to explore the affinity of immunoglobulins and immunoglobulin fragments for the mast cells of the mouse tongue. Human IgG but not IgA or IgM showed such an affinity as revealed by fluorescence of globulin attached to the mast cell surface and to the mast cell granules. This affinity was found also in isolated H(γ) chains but not in the light chains of IgG. After papain digestion, the Fab and Fc fragments obtained showed no affinity for mouse tongue mast cells though the 5S fragment obtained after pepsin digestion retained activity. The human immunoglobulin sub-class IgG₂ lacks the ability to bind to mouse tongue mast cells. In guinea-pig serum, γ₁- but not γ₂-globulin showed an affinity for mouse tongue mast cells. It was suggested that a specific attachment site for mouse tongue mast cell surface receptors was present on the γ chain of human IgG and that this site was in a position susceptible to attck by papain hydrolysis.     

Total immunoglobulin of rat thymocytes and thoracic duct lymphocytes

The total immunoglobulin of rat thoracic duct lymphocytes (TDL) and thymocytes has been assayed in extracts of detergent-solubilized cells. Immunoglobulin was found in thymocytes in much larger amounts than expected from cell surface assays. The main classes involved were IgA and IgG_2a+2b, with very little IgM being detected. The variability in the amount detected and the increase with age suggested that the immunoglobulin was coming from antibody-forming cells in the thymus. TDL contained large amounts of internal IgA, but virtually no hidden IgG_2a+2b or IgM. The IgA was in large lymphocytes which also had surface immunoglobulin. Most of the IgM in cell extracts seemed to be derived from the cell surface. It was not found in amounts greater than estimated by cell surface assays, and was confined to small lymphocytes which carried large amounts of cell surface immunoglobulin. Less than 1000 molecules per cell of IgM was detected in thoracic duct lymphocytes with little surface immunoglobulin after they had been solubilized in 0.33 % sodium dodecyl sulfate, 1 % Triton X-100 or 9 M urea + 1.5 M acetic acid.     

Roles of follicular helper and regulatory T cells in allergic diseases and allergen immunotherapy

Allergic diseases are characterized by overactive type 2 immune responses to allergens and immunoglobulin E (IgE)-mediated hypersensitivity. Emerging evidence suggests that follicular helper T (T_FH) cells, rather than type 2 T-helper (T_H2) cells, play a crucial role in controlling IgE production. However, follicular regulatory T (T_FR) cells, a specialized subset of regulatory T (T_REG) cells resident in B-cell follicles, restricts T_FH cell-mediated help in extrafollicular antibody production, germinal center (GC) formation, immunoglobulin affinity maturation, and long-lived, high-affinity plasma and memory B-cell differentiation. In mouse models of allergic asthma and food allergy, CXCR5⁺ T_FH cells, not CXCR5⁻ conventional T_H2 cells, are needed to support IgE production, otherwise exacerbated by CXCR5⁺ T_FR cell deletion. Upregulation of T_FH cell activities, including a skewing toward type 2 T_FH (T_FH2) and IL-13 producing T_FH (T_FH13) phenotypes, and defects in T_FR cells have been identified in patients with allergic diseases. Allergen immunotherapy (AIT) reinstates the balance between T_FH and T_FR cells in patients with allergic diseases, resulting in clinical benefits. Collectively, further understanding of T_FH and T_FR cells and their role in the immunopathogenesis of allergic diseases creates opportunities to develop novel therapeutic approaches.     

Fine‐needle aspiration of tubulocystic renal cell carcinoma

We report two cases of tubulocystic renal cell carcinoma, a rare renal tumor the cytology of which has not been previously reported. Both aspirates were cellular and contained large sheets of cells with abundant granular cytoplasm, distinct cell borders and intracellular windows, distinct to prominent nucleoli, rare intracytoplasmic vacuoles, and rare nuclear grooves. Cells with variable amounts of cytoplasm were also arranged in small groups, some of which resembled spherules. The large sheets of cells with windows appeared specific for tubulocystic carcinoma; the spherules could easily be confused with a papillary renal cell carcinoma.     

Use of an automatic cell harvester in a cellular radioimmunoassay

An automatic cell harvester was used in the final step of a cellular radioimmunoassay to collect cell bound anti-rat IgG ¹²⁵I-F(ab′)₂. Studies on the reliability of this collection method were performed with antibodies directed against cell surface antigens induced by the Gross murine leukemia virus and produced by immunization of W/Fu rats with the syngeneic (C58NT)D lymphoma. Glutaraldehyde-fixed as well as untreated Gross virus induced lymphoma cells could be used. Similar and specific antibody binding curves were obtained when the cells were incubated with the anti-(C58NT)D serum and anti-rat IgG ¹²⁵I-F(ab′)₂ in the presence of 0.1% NaN₃. Background levels of non-specific binding of anti-rat ¹²⁵I-F(ab′)₂ to mouse lymphoma cells or rat thymocytes were only a few cpm above the background of the gamma-counter. This allowed detection of surface immunoglobulin positive lymphocytes among as few as 30,000 rat splenocytes. In addition, this cellular radioimmunoassay was found to be suitable for the measurement of solubilized cell surface antigen by its capacity to inhibit binding of the specific antibodies to the target cells.     

Antigen and helper T lymphocytes activate B lymphocytes by distinct signaling pathways

Resting murine B lymphocytes can be induced to proliferate by cross-linking membrane immunoglobulin, the antigen receptor, or by contact with activated helper T lymphocytes in the absence of a signal through membrane immunoglobulin. Little is known about the molecular nature of contact-dependent T cell help. To determine whether helper T cells activate B cells through different signal transduction and second messenger pathways from those used by membrane immunoglobulin, the effects of drugs which block activation of B cells through membrane immunoglobulin were measured on B cell activation by contact with anti-CD3-activated and fixed T helper cells. Cyclosporin A, phorbol esters added at the time of activation, and cAMP agonists all block activation of B cells through membrane immunoglobulin at concentrations at least 100-fold lower than those necessary to block B cell activation by contact with activated T_h1 or T_h2 helper T cells. Depletion of protein kinase C by pretreatment of B cells with phorbol ester inhibits the proliferative response to anti-immunoglobulin but not the response to contact with activated T cells. The B cell response to lipopolysaccharide is intermediate in sensitivity to cyclosporin A and cAMP agonists, and resembles the response to activated T cells in resistance to phorbol esters and protein kinase C depletion. Various protein kinase inhibitors did not distinguish among these B cell activation pathways, except for the tyrosine kinase inhibitor, herbimycin A, which inhibited anti-immunoglobulin responses at 3- to 5-fold lower concentrations.     

A seroreactive 120-kilodalton beta-1,3-glucanase of Coccidioides immitis which may participate in spherule morphogenesis.

A beta-glucosidase of Coccidioides immitis was identified in electrophoresis gel separations of the concanavalin A-bound mycelial culture-filtrate-plus-lysate preparation. p-Nitrophenol-beta-D-glucopyranoside was used as the substrate to visualize the enzymatically active fraction in nonreducing gels. The gel-isolated, chromatographically purified enzyme has an optimal pH of 8.0 and cleaves beta-1,3-glycosyl linkages. The alkaline beta-glucosidase was further characterized by a pI of 3.8 to 4.0, optimal activity at 37 to 40 degrees C, and molecular size of 120 kDa as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified beta-glucosidase is identical to a previously reported 120-kDa antigen (Ag) which reacts with immunoglobulin M (IgM) tube precipitin (TP) antibody in sera from patients with coccidioidomycosis. The TP-Ag was described as a valuable serodiagnostic reagent for detection of specific IgM in patients with early coccidioidal infections. The beta-glucosidase, like the TP-Ag, was localized in the cell wall and cytoplasmic vesicles of parasitic cells (spherules) by immunofluorescence and immunoelectron microscopy with specific antiserum raised against the purified enzyme. The boiled cell wall fraction isolated from these same young (presegmented) spherules was partially digested by the beta-glucosidase. Addition of a potent beta-glucosidase inhibitor, 1-deoxynojirimycin, to the parasitic-phase culture medium at a concentration of 200 microM blocked or retarded conversion of arthroconidia to spherules. Antibody was raised in guinea pigs against chromatographically purified 1-deoxynojirimycin which was conjugated with bovine serum albumin. The inhibitor was localized by immunofluorescence in the wall of the 1-deoxynojirimycin-treated cells. We suggest that the spherule wall-associated, alkaline hydrolase functions as a beta-1,3-glucanase to provide for wall plasticity as well as intussusception of newly synthesized wall polymers during the period of rapid diametric growth of parasitic cells of C. immitis.     

Activation of T and B cells by a crude extract of Artocarpus integrifolia is mediated by a lectin distinct from jacalin

The biological activities previously described for a crude extract derived from seeds of Artocarpus integrifolia (jack fruit) are shown in the present work to be assigned to two distinct fractions present in this extract. One fraction is the -galactose binding lectin, jacalin, obtained by affinity purification on a -galactose Sepharose column. The other fraction is a -mannose-binding protein which we propose to call ‘Artocarpin’. As is well documented, jacalin binds to human IgA₁ and is a useful tool for the purification of this immunoglobulin. We show here that the remaining biological activities consisting of the proliferative response of mouse spleen cells and human peripheral blood mononuclear cells and polyclonal activation of human and mouse B cells for the secretion of immunoglobulin are mediated by artocarpin. Artocarpin is unique in its capacity to induce polyclonal activation of B cells in the absence of proliferation. BALB/c nu/nu spleen cells failed to proliferate which indicates that this lectin is a T cell-dependent B cell polyclonal activator.     

Characterization of mouse thoracic duct B lymphocytes I. Evidence of functional heterogeneity

Thoracic duct lymphocytes (TDL) from C 3 H/Tif and BALB/c mice were studied for their in vitro reactivity to the B cell mitogens lipopolysaccharide (LPS) and lipoprotein (LP). Roughly 4% and 10% of the surface immunoglobulin (Ig)-positive cells in these populations could be stimulated by LPS and LP, respectively, to generate clones of IgM-secreting cells. Among LPS-reactive B cells, roughly 30% developed into clones which also produced IgG₃ or IgG₂, while only a very small fraction (1–2%) of all precursors could give rise to clones secreting IgG₁ and IgA. Freshly collected TDL from some batches of C 3 H/HeJ mice displayed a high proportion of Ig-containing B cell blasts (5–10%), which did not secrete enough Ig to be detected as plaque-forming cells (PFC). These cells, however, under appropriate culture conditions and stimulated by LP (but not by Nocardia mitogen), differentiated to PFC of the various Ig classes without dividing.