Secretory component of epithelial cells is a surface receptor for polymeric immunoglobulins.

Epithelial cells of human fetal intestines and of a colonic carcinoma cell line (HT-29) exhibited intracellular and surface binding of polymeric immunoglobulins of IgA and IgM classes; monomeric IgA and IgG did not bind to these cells. Secretory component was identified as the receptor involved in the immunoglobulin binding. This conclusion was confirmed by the following experiments: trypsin abrogated the surface binding of polymeric immunoglobulin, reappearance of surface secretory component (SC) restored immunoglobulin binding; the appearance of SC in developing fetal tissues coincided with their potential to bind polymeric immunoglobulin; anti-SC reagents inhibited the binding of immunoglobulins to epithelial cells; and SC-containing secretory IgA did not bind to the surface of HT-29 cells.

Characterization of gastric mucosal membranes. X. Immunological studies of gastric (H+ + K+)-ATPase.

Gastric mucosal homogenates from hog were fractionated by differential and density gradient centrifugation and free-flow electrophoresis. The two major membrane fractions (FI and FII) thus obtained are distinct both enzymically and in terms of transport reactivity. This heterogenicity extends to their antigenic activity. Purified antibodies which were raised against the K+-ATPase-containing H+ transport fraction FI were of two types: inhibitory and non-inhibitory. Inhibitory antibodies reduced the K+-ATPase activity by approximately 80% and the K+-p-nitro-phenylphosphatase activity by approximately 40% in a concentration-dependent manner, while the small Mg++-dependent component of the enzyme activity was unaffected. Antibodies inhibiting the K+-ATPase also inhibited H+ transport. These antibodies did not cross-react with the other major membrane fraction isolated by free-flow electrophoresis, FII, and gave a single band on rocket immunoelectrophoresis. Antibodies against this FII fraction also did not react with the K+-ATPase and were heterogeneous, giving at least four bands with rocket immunoelectrophoresis and inhibiting both the 5'-nucleotidase and Mg++-ATPase of this fraction. Immunofluorescent staining of tissue sections showed that the FI was derived from the parietal cell of gastric tissue and was localized to the supranuclear area of the cell. Staining of isolated rat gastric cell suspensions by FI antibodies confirmed the selectivity of the antibody and showed a polar, plasma membrane localization. FII antibodies also largely stained the parietal cells in tissue sections. In the 16 hog tissues tested, FI antibodies cross-reacted only with gastric fundus, thyroid and weakly with thymus. Immunoelectronmicroscopy showed that FI antibodies reacted strongly with the secretory membrane at the apical cell surface of the parietal cells and at the secretory canaliculi, weakly with the apical surface of the zymogen cell, and not with the basal-lateral surface of the cells. Thus, the protontranslocating ATPase is localized in the parietal cells and in the region postulated to be the site of acid secretion.

An acid transporting enzyme in human gastric mucosa.

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.

Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies.

Circulating immune complexes (CIC) containing IgA and C3 were elevated in 48% of IgA nephropathy patients; IgA1 was the predominant subclass. IgA1-IgG CIC were detected in 44%, IgA2-IgG CIC in 7%, and IgM-IgA1 CIC in 16% of the patients. No IgM-IgA2 CIC were detectable. Sucrose gradient ultracentrifugation indicated that IgG-IgA1 CIC were predominantly of intermediate (13-19S) size whereas IgA1-C3 CIC sedimented from 11S to 19S. At acid pH, isolated CIC revealed the presence of substantial amounts of 7S IgA. One third of the patients had elevated serum IgA rheumatoid factor (RF) of both polymeric and monomeric forms despite normal levels of IgM-RF; 87% of patients with elevated IgA-RF had IgA1-IgG CIC. These results indicate that the IgA1 component of CIC in patients with IgA nephropathy is not necessarily of mucosal origin and suggest that a portion of these CIC consists of IgA RF immunologically complexed with autologous IgG.

A comparative analysis of cytoplasmic mu (C mu) expression in acute lymphoblastic leukemia by molecular and immunologic techniques. Identification of leukemia cases expressing C mu mRNA transcripts in the absence of detectable C mu proteins.

Among acute lymphoblastic leukemias derived from the B-cell lineage, the subset of cases expressing cytoplasmic mu heavy chain proteins (C mu) in the absence of surface immunoglobulin has been designated pre-B-cell acute lymphoblastic leukemia. This group, traditionally identified using immunologic smear techniques, has been associated with a poor prognosis in some series. In a comparative study, 25 cases of B-lineage acute lymphoblastic leukemia were analyzed for C mu expression using molecular and immunologic techniques. RNA derived from cryopreserved blast cells was hybridized in both Northern and slot-blot analyses using a probe (pBZ311) containing four exons of the human immunoglobulin heavy chain mu constant region gene. Expression of C mu proteins was assessed simultaneously by slide immunofluorescence and flow cytometric techniques in all samples. These studies were correlated with immunoglobulin heavy and light chain gene rearrangements, cell-surface immunophenotype, cytogenetics, and other clinicopathologic features. C mu mRNA transcripts were detected in 14 of 25 cases, whereas C mu proteins were detected in only 9 of these cases using flow cytometric techniques. Only four of these nine cases were positive by slide immunofluorescence techniques. These studies imply that molecular and flow cytometric techniques may be a more sensitive means to assess C mu expression. The identification of five cases that expressed C mu mRNA transcripts in the absence of detectable C mu proteins also suggests that molecular techniques may be valuable in identifying a unique subgroup of pre-B-cell acute lymphoblastic leukemia cases that contain C mu mRNA transcripts, but lack C mu proteins.

Organization and operation of a flow cytometric immunophenotyping laboratory.

Technical advances in the field of flow cytometry have made it feasible for many academic and private hospital laboratories to purchase relatively inexpensive "user friendly" flow cytometers that do not require dedicated flow cytometer operators, special rooms, or a significant amount of laboratory space. Because the financial and physical constraints in operating a flow cytometer have been substantially reduced, many pathologists may now be considering such a purchase. By chronicling the clinical activities of a single flow cytometric immunophenotyping laboratory, this report will answer a variety of questions that may be asked by pathologists regarding both the utility of flow cytometers in clinical diagnosis and the mechanics of operating an immunophenotyping laboratory. The types of tissues that can be evaluated by flow cytometry will be detailed, and we will summarize the number and type of flow cytometric clinical studies performed in our laboratory since its development in 1983. Practical aspects of laboratory operation including technical staff requirements, specimen handling and processing procedures, monoclonal antibody selection, and quality control procedures will be presented. In addition, a comprehensive review of flow cytometric immunophenotyping studies as applied to the diagnosis of leukemias, lymphomas, and immunodeficiency disorders will be presented along with case examples that illustrate our approach to the interpretation of immunophenotyping results.

Flow cytometric analysis of lymphoma and lymphoma-like disorders.

The use of flow cytometry (FC) represents the most recent advance in the phenotypic analysis of lymphocyte subsets, and has emerged as a valuable adjunct in the diagnosis of malignant non-Hodgkin's lymphoma (NHL). In a review of over 200 cases of nodal and extranodal suspected lymphomas studied in the Immunophenotyping Laboratory at the University of New Mexico, the diagnostic utility of FC was assessed. Among cases of NHL, FC was able to confirm a morphologic diagnosis of lymphoma and determine B or T cell lineage in greater than 85% of the samples submitted. Difficulty in lineage determination in the remaining cases of morphologic NHL was multifactorial. Among cases of reactive lymph nodes and Hodgkin's disease, FC showed no characteristic patterns, although several cases exhibited phenotypic profiles suggestive of B or T cell clonality. When combined with routine morphologic review and accompanied by other specialized diagnostic techniques when necessary, the use of FC represents a precise and reproducible method for rapidly and easily studying lymphoproliferative disorders in solid tissue.

Molecular-cellular interactions in the secretory IgA system.

1) SC receptors were not detected on the surface of human PBL before or after PWM stimulation or on the surface of established lymphoblastoid cell lines. 2) SC binding was detected in the cytoplasm of differentiated lymphoid cells. The majority of the SC-binding cells contained intracellular IgA. 3) The binding of polymeric IgA to the surface of human epithelial cells (colonic carcinoma HT-29) was dependent on the presence of SC. 4) These findings indicate that SC is a receptor and possible transport protein for polymeric immunoglobulins, but that it is not directly involved in the homing of the IgA precursor cells to secretory tissues.

Concept of the local and common mucosal immune response.

The available information concerning the origin of IgA-producing plasma cells and the spectrum of IgA-associated antibodies found in external secretions provide arguments that support two pathways of stimulation for a secretory humoral immune response. In addition to an explicitly local immune response induced by a topical antigen application, a second mechanism of induction operating through the sensitization of GALT, and possibly BALT, emerges. The latter pathway of stimulation leads to the appearance of specific IgA-associated antibodies in secretions of mammary, salivary, and lacrymal glands (and perhaps other sites), all of which suggests the existence of a common mucosal secretory system. Not yet explained are the mechanisms involved in the homing to secretory glands of cells sensitized in the remote lymphoid tissues (GALT and BALT), the differentiation patterns of these cells, and the regulation of selective IgA expression.

Common problems in the diagnosis of immunodeficiency in children.

Primary immunodeficiency (ID) has an incidence of 1 in 10,000 and may have significant morbidity and mortality if not diagnosed and treated early. Children with signs and symptoms suggestive of immunodeficiency are often seen first by their paediatrician or family medical practitioner. For these reasons, it is essential that primary care physicians both of children and adults should be able to recognize the signs and symptoms of immunodeficiency and be knowledgeable about the screening procedures useful in the diagnosis of these diseases. However, this process is often complicated by the diverse presentations of immunodeficiency and the lack of specificity and relative inaccessibility of screening tests. The purpose of this paper is to address the major problems in diagnosis of immunodeficiency, including: 1) Which patients to seriously consider for diagnosis? 2) How to begin the work-up for primary immunodeficiency? 3) Which procedures are useful in the diagnosis of the various forms of immunodeficiency? and 4) What is the relative importance of immunoglobulin (IgG) subclasses?

Human colostral cells. II. Response to mitogens.

The ability of colostral lymphocytes to respond to pokeweed mitogen, phytohemagglutinin, or Epstein-Barr virus was examined. None of these mitogens induced colostral cells to differentiate into immunoglobulin-containing cells, either in the absence or in the presence of mitomycin C-treated mononuclear cells or T-cell-enriched populations from peripheral blood. Cocultivation of mononuclear cells from the peripheral blood of normal adults with mitomycin C-treated colostral cells resulted in a marked suppression of the generation of immunoglobulin-containing cells in response to pokeweed mitogen. The inhibitory effect was seen in peripheral blood mononuclear cell:colostral cell ratios of 1:1, 5:1, and 10:1. However, colostral cells had little effect on the ability of peripheral blood lymphocytes to proliferate in response to phytohemagglutinin or to allogeneic stimulation.

Secretory component: interactions with intracellular and surface immunoglobulins of human lymphoid cells.

Unstimulated and PWM-stimulated lymphocytes from normal human peripheral blood, cord blood, peripheral blood of patients with panhypogammaglobulinemia and selective IgA deficiency, as well as human lymphoblastoid cell lines were examined for their ability to bind secretory component (SC) on the surface and in the cytoplasm. SC binding was not detected on the cell surface at any stage of differentiation in these cells. However, binding of SC was detected in the cytoplasm of 2.3% of normal peripheral blood lymphocytes cultured in the presence of PWM for 6 to 7 days, and in two IgA producing lymphoblastoid cell lines. The capability of lymphoid cells to bind SC was not concurrent with J chain production. Although IgA was detected in the cytoplasm of PWM-stimulated lymphocytes from IgA-deficient patients, these cells did not bind SC. The failure to detect surface receptors indicates that SC is not a probable factor determining the homing of IgA precursor cells into exocrine tissues.

Immunohistochemical distribution of immunoglobulins, lactoferrin, and lysozyme in human minor salivary glands.

The immunofluorescence technique was used to examine the distribution of immunoglobulin A and its subclasses, secretory component (SC), J chain, lactoferrin and lysozyme in labial and lingual (von Ebner's) glands. IgA-containing plasma cells were found in the connective tissue around intercalated or intralobular ducts and a few were noted around acini of both glands. IgA was detected in the apical cytoplasm of intercalated and intralobular duct cells and in acini of von Ebner's glands and in demilunes of labial glands. Most IgA-containing cells also stained for J chain. The ratio of IgA1:IgA2-containing cells was approximately equal in von Ebner's and labial glands. Cytoplasmic and surface membrane-related staining for SC was detected in epithelial cells of the intercalated and intralobular ducts in both glands, in the serous acini of von Ebner's gland, and in the demilunes of labial glands. Lactoferrin was found in serous acini, demilunes, intercalated and intralobular ducts. Lysozyme was found in acinar and intercalated ducts, but was rarely seen in intralobular ducts. These results disclose the presence of cells (plasma cells and epithelial cells) and their products (IgA and secretory component) that indicate the local production of secretory IgA in minor salivary glands.

Regulation of IgA expression by isotype-specific T cells and soluble binding factors.

The data presented here suggest a model for isotype-specific regulation of IgA synthesis by Fc alpha R+ T cells (Figure 1). Immature mIgM+ +/- mIgD+ B cells are induced by T switch cells to express cell surface IgA (a phenotypic switch). If the T switch cell induces mIgA expression via a long primary RNA transcript from an unrearranged C alpha allele, the hypothetical intermediate switch B cell results (step 1); this may be the mechanism of heavy chain expression in memory B cells that express low levels of Ig. Alternatively, T switch cells may induce a DNA rearrangement in the CH locus of the B cell (a genotypic switch), which results in a deletion of all CH loci except C alpha (step 2). TH inducer cells promote maturation of mIgA+ B cells to IgA-secreting plasma cells. This may involve a DNA switch rearrangement (step 3) or the maturation of previously switched cells (step 4), and appears to be mediated via an IgABF with enhancing activity. Not shown in this figure, but inherent in this model, is a suppressive regulatory arm that may be mediated via IgABF with suppressive activity released from Fc alpha R+ suppressor T cells. Due to the presence of Fc alpha R on a variety of cell types, IgABF may suppress synthesis of IgA by acting not only on mIgA+ B cells but also on regulatory cells (T cells, B cells, and macrophages) bearing IgA bound to Fc alpha R. If the IgA system is analogous with the IgE system, mIgA-bearing B cells may be the direct target of IgABF. Binding of Ig to FcR has been shown to (a) increase the number of Fc receptors per cell, (b) enhance the number of cells expressing Fc receptors, (c) induce the release of IgBF that either suppress or enhance Ig secretion, and (d) effectively convert surface Ig- cells into surface Ig+ cells that are therefore receptive to IgBF. Thus, FcR+ cells may interact with IgBF and Ig via a regulatory network to stimulate or inhibit the immune response in an isotype-specific manner. Cell surface molecules (mIg, FcR) may serve as sensors that allow the cell to detect and respond to fluctuations in the levels of immune mediators that serve to modulate Ig synthesis and secretion. The relationship between IgBF and FcR is not known, nor is it known whether Fc receptors expressed by different cell types are encoded by the same gene and are controlled similarly.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of IgA and IgM in human colostral elements using immunoelectron microscopy.

In addition to the free form, IgA is associated with cellular and noncellular elements present in human colostrum. To resolve the existing controversy as to the cell type(s) containing IgA, we used immunoelectron microscopy with horseradish peroxidase-labeled F(ab')2 or Fab' fragments of anti-IgA or anti-IgM to determine the distribution of these immunoglobulins in colostral elements. IgA and IgM were localized in phagocytic vacuoles of polymorphonuclear leukocytes and macrophages in the vicinity of the cell membrane. In neutrophilic leukocytes, both immunoglobulins were occasionally found in phagocytic vacuoles distributed throughout the cytoplasm. Although the in vitro phagocytic activity of colostral cells was low, they retained the ability to ingest colloidal gold particles which were subsequently localized in phagocytic vacuoles that also contained IgA or IgM. IgA and IgM were not detected in lymphocytes, and plasma cells were not found in human colostrum. Numerous noncellular colostral globules of various shapes and sizes also contained IgA and IgM. These observations indicate that IgA and IgM were acquired by phagocytic cells and noncellular globules and were not actively synthesized by lymphoid cells present in human colostrum.