The value of immunofluorescence in the study of human renal disease.

Immunofluorescence is a useful technique in the study of human renal diseases, both from the point of view of elucidating pathogenic mechanisms and as a diagnostic tool. The finding of characteristic staining patterns for immunoglobulins and complement indicates that many forms of glomerulonephritis are immune complex diseases and that a few are due to anti-GBM antibodies. On the other hand, in lipoid nephrosis and toxemia of pregnancy, deposits of immunoglobulins and complement are generally absent, indicating that immunologic mechanisms are probably not responsible for these glomerular diseases. The finding of fibrin or other fibrinogen derivatives in glomeruli in toxemia of pregnancy and in certain forms of glomulonephritis supports the interpretation that these substances play a pathogenic role in certain glomerular diseases. The use of immunofluorescence has led to the recognition of two previously unrecognized renal diseases: nephropathy with mesangial IgA-IgG deposits (Berger), and a tubular disorder with deposits of immunoglobulins and complement along the basement membrane.

The nature and the specificity of mononuclear cells in experimental autoimmune inflammations and the mechanisms leading to their accumulation.

The nature and specificity of the mononuclear cells in passively transferred autoimmune encephalomyelitis and adrenalitis were studied. The recipients were prepared by production of a small heat lesion in the target tissue 5 days before transfer. Within 24 hr after transfer of lymph node cells from donors sensitized with the corresponding tissue antigen, a dense mononuclear cell infiltrate developed around the lesion. When lymph node cells labeled in vitro with (3)H-thymidine or (3)H-adenosine were transferred, a significant number of labeled lymphocytes was found in the infiltrate at 24 or 48 hr. Lymphocytes labeled with (3)H-thymidine showed a greater tendency to accumulate than cells labeled with (3)H-adenosine, indicating that newly formed lymphocytes were more prone to enter the reaction than older cells. Labeled lymphocytes and macrophages of recipient origin and labeled lymphocytes from donors stimulated with B. pertussis were also shown to accumulate around the heat lesion provided the reaction had been initiated by transfer of unlabeled lymphocytes from donors sensitized to the appropriate tissue-specific antigen. In recipients which were given lymph node cells from two groups of donors, sensitized either to spinal cord or to adrenal antigens, with cells from only one group of donors labeled, equal percentages of labeled cells were found around each lesion. Thus, no evidence of preferential accumulation of specifically sensitized lymphocytes was obtained. In recipients which received whole body irradiation on the day of production of the heat lesions, 5 days before transfer of lymph node cells from appropriately sensitized donors, neither monocytes nor lymphocytes accumulated around the lesion. However, if the tibial bone marrow was shielded or if bone marrow cells were given to the recipients shortly after irradiation, inflammation developed as in normal recipients. In recipients which were irradiated 24 hr after the transfer of unlabeled lymph node cells from donors sensitized to the appropriate tissue antigen and then given labeled lymph node cells from B. pertussis-stimulated donors, labeled lymphocytes were found in the reaction 24 hr later. This accumulation occurred although virtually all the lymphocytes present in the lesion at 24 hr after the first transfer were destroyed by the irradiation. The results are interpreted as follows. The autoimmune reaction is initiated by the arrival at the site of a few specifically sensitized lymphocytes, probably on a random basis. After contact with antigen, factors are produced and released which cause the influx of monocytes and of lymphocytes, in particular newly formed ones, of various specificities. There is no preferential accumulation of specifically sensitized cells. The influx of lymphocytes appears to require the presence of monocytes or macrophages in the reaction.

The distribution of large dividing lymph node cells in syngeneic recipient rats after intravenous injection.

The distribution of large dividing lymph node or thoracic duct lymph cells, labeled in vitro with (3)H-thymidine, was studied in syngeneic recipient rats after intravenous injection. In most experiments the donor rats had been immunized with Bacillus pertussis 4 days earlier, but in some instances cells from nonimmunized donors were used. In smears, the labeled donor cells had the appearance of large lymphocytes or large pyroninophilic cells. By electronmicroscopy, the majority of labeled donor cells were seen to have only scanty endoplasmic reticulum. It was found that the labeled cells rapidly "homed" to lymphoid tissue and recirculated in the recipient, in a fashion resembling that of small lymphocytes. However, the distribution of labeled cells was found to depend upon the source of the donor cells. Cells from mesenteric lymph nodes or thoracic duct lymph showed a marked preferential accumulation in lymphoid tissue within or adjacent to the intestine, whereas cells from peripheral nodes accumulated preferentially in peripheral lymph nodes. Cells from any of these sources showed an equal tendency to accumulate in the white pulp of the spleen. Suspensions of small lymphocytes, labeled in vitro with (3)H-uridine, did not display a similar tendency to localize preferentially in lymphoid tissue in certain regions. It was also found that large dividing lymph node cells from donors immunized with an antigen (2,4-dinitrophenyl-bovine gamma globulin (DNP-BGG) or B. pertussis) showed a greater tendency to accumulate in a recipient lymph node containing that antigen than in the contralateral node. It was not determined whether the selective accumulation of large dividing lymphoid cells from different sources in lymphoid tissue of different regions in recipients was due to an antigen recognition mechansim or was the result of two different populations of cells with different "homing" mechanisms.

Studies of PPLO infection. I. The production of cerebral polyarteritis by Mycoplasma gallisepticum in turkeys; the neurotoxic property of the Mycoplasma.

Turkey poults injected intravenously with suspensions of Mycoplasma gallisepticum develop a fatal neurologic disease associated with polyarteritis affecting almost exclusively the cerebral arteries. The incubation period depends on the dose of organisms. With high doses (10(10) to 10(11) mycoplasmas) the birds become ill and die within a few hours; with lower doses (10(6) to 10(8)) neurologic manifestations appear after 7 days. The rapid onset of neurologic signs after high doses indicates the presence of a toxin in the mycoplasma, but efforts to extract toxin from disrupted organisms or to demonstrate its presence in culture fluid free of mycoplasmas have been unsuccessful. The toxin appears to be associated only with living mycoplasmas. The toxic component of M. gallisepticum is inactivated by heating the organisms at 50 degrees C, disruption by repeated cycles of freezing and thawing, and exposure to specific antibody. Treatment of turkeys with gold thiomalate furnishes partial protection against the toxic effects of large doses of mycoplasmas, and protection against the development of cerebral arteritis. Treatment with tetracycline protects completely against both toxicity and arteritis, and, when delayed, restores diseased birds to a healthy state. Cortisone, methotrexate and 6-mercaptopurine have no effect on the course or outcome of the disease. Intracerebral injections of M. gallisepticum are less toxic and lethal than when the same dose was given by vein, indicating that the organism exerts its damaging action on blood vessels by way of the blood stream. The arterial lesions resemble those of serum sickness, except for their distribution, and are associated with glomerular inflammatory lesions. However, for various reasons discussed, it is considered more likely that they result from a direct toxic action of living mycoplasmas on the vessels concerned than from an immunologic mechanism.

Passive transfer of autoimmune disease with isologous IgG1 and IgG2 antibodies to the tubular basement membrane in strain XIII guinea pigs: loss of self-tolerance induced by autoantibodies.

Initiation of an autoimmune tubulointerstitial disease was achieved in strain XIII guinea pigs by passive transfer of functionally pure IgG1 or IgG2 fractions of isologous anti-tubular basement membrane (TBM) serum. IgG2 appeared to be somewhat more effective than IgG1. The immunopathologic features in the IgG1 and IgG2 recipients were similar at the time of sacrifice, 14 days after transfer. The recipients that developed disease had higher than expected anti-TBM titers at 14 days. Furthermore, anti-TBM antibodies were of both IgG isotypes. In contrast, simultaneously administered IgG1 or IgG2 anti-BGG antibodies declined in titer in the recipients and were never found in the isotype fraction that had not been transferred. These findings indicate that the recipients of anti-TBM antibodies of either IgG1 or IgG2 isotype were stimulated to produce anti-TBM autoantibodies, which participated in the pathogenesis of the renal disease. The model demonstrates that autoantibodies may provide a mechanism (autoimmune amplification) for the intensification and perpetuation of antibody-mediated autoimmune diseases.

The bacterial induction of homograft sensitivity. 3. Effects of group A streptococcal membrane antisera.

Immunization of rabbits with Group A Type 12 streptococcal cell membranes has elicited serum antibodies which have the ability to cause rapid rejection of skin allografts in guinea pigs. Intradermal injection of such antisera has resulted in skin reactions characterized by prominent polymorphonuclear leukocyte infiltrates, similar to those noted in the Arthus reaction. The combined use of membrane antisera and epinephrine has resulted in hemorrhagic necrosis of the skin of guinea pigs. The ability of membrane antisera to exert these effects appears to be dependent upon the presence in the host tissues of antigen(s) shared by or cross-reacting with streptococcal membrane antigens. Such cross-reacting antigens may have a group distribution in the outbred guinea pig population. The results highlight the potential biological importance of antigens present in the Group A streptococcal membrane in the induction of altered tissue reactivity in the mammalian host.

T-cell migration into allografts.

The ability of T and B lymphocytes to migrate into skin allografts undergoing rejection was studied in mice. Spleen cells from CBA/J mice sensitized to transplantation antigens of A/J or C57BL/6 mice were separated on immunabsorbent columns into purified populations of T and B cells, labeled in vitro with 3H-uridine and injected intravenously into CBA/J mice with 7-day old skin iso and allografts (A/J or C57BL/6). The mice were sacrificed 24 h later and studied by autoradiography. After transfer of either unfractionated spleen cells or T cells, large numbers of labeled cells were found in the cellular infiltrate of allografts, whereas extremely few were seen in isografts. In contrast, after transfer of B cells, almost no labeled cells were detected either in the allografts or the isografts, although they, like T cells, homed normally to lymphoid tissue.

A cell-mediated reaction against glomerular-bound immune complexes.

Lewis rats were given a single i.v. injection of soluble immune complexes containing human serum albumin (HSA) and rabbit anti-HSA antibodies, prepared in antigen excess. This resulted in localization of HSA and rabbit gamma globulin (RGG) in glomerular mesangial regions without producing definite histologic changes. 24 h after the injection of immune complexes, groups of these rats received lymph node cells or T-cell preparations from syngeneic donors sensitized to RGG, HSA, or ovalbumin; another group received no cells. All of these groups and a group of normal control rats were given injections of [3H]thymidine at 18, 27, and 44 h. The animals were killed 48 h after the time of cell transfer. In histologic sections, glomerular abnormalities were found only in some of the animals that had received immune complexes and lymph node cells or T-cell populations from donors sensitized to HSA or RGG; the lesions were characterized by focal and segmental increase in cells in mesangial regions. Autoradiographs revealed significantly greater numbers of labeled cells in mesangial regions and glomerular capillaries in the groups that had received immune complexes and cells from HSA- or RGG-sensitized donors than in any of the other groups. Electronmicroscopic studies suggested that the increase in cellularity in mesangial regions resulted from an influx of mononuclear phagocytes. The findings indicate that cell-mediated reactions can be initiated by the interaction between sensitized T lymphocytes and antigens present in immune complexes within mesangial regions.

Evidence for a pathogenic role of a cell-mediated immune mechanism in experimental glomerulonephritis.

Lewis rats were injected intravenously with rabbit anti-rat glomerular basement membrane (GBM) antisera in doses that were sufficient to cause glomerular fixation of rabbit gamma globulin (RGG) detectable by immunofluorescence, but which failed to induce histologically detectable lesions. 24 h later, groups of rats received lymph node cells or serum from syngeneic donors that had been immunized with either RGG or ovalbumin; they were injected with [3H]thymidine three times during the next 2 days, and sacrificed 48 or 96 h after transfer. Only the rats given anti-GBM antiserum plus lymph node cells from donors sensitized to RGG showed histological glomerular lesions, in the form of segmental hypercellularly and necrosis. Autoradiographs revealed the greatest number of labeled cells in glomeruli in the same group. In analogous experiments, it was shown that T-cell-enriched populations could induce hypercellular glomerular reactions. On the basis of electronmicroscopic and autoradiographic observations, it appears that the glomerular hypercellularity resulted from both infiltration of mononuclear cells and proliferation of endothelial cells. The findings indicate that interaction of specifically sensitized lymphocytes with glomerular-bound antigen can induce a cell-mediated (delayed-type) reaction in glomeruli.

Immunological effects of thermal injury. I. Inhibition of spermatogenesis in guinea pigs.

PIP: In the present study, controlled burns of 1 testis have induced the formation of testis-specific antibodies in Hartley guinea pigs. The antibodies reacted with autologous as well as homologous testicular extracts. In addition, pathological changes have been noted in the germinal components of the contralateral testis, which were similar to those observed after the induction of experimental allergic orchitis by active immunization with testicular tissue. Results of the study indicate that thermal injury to guinea pig testes can induce an autoimmune response similar to that observed after immunization with autologous or homologous testicular tissue. The antibodies formed were organ-and species-specific against a testicular antigen. Thermal injury may be associated with autoimmunization of the host by the injured organ.^ieng

Altered functional properties of the renal glomerulus in autologous immune complex nephritis: an ultrastructural tracer study.

The altered functional properties of the glomerular capillary wall in a model of autologous immune complex disease (Heymann's nephritis) was studied by electron microscopy using intravenously injected protein tracers of varying molecular weight. There was an increase in the permeability of the glomerular basement membrane (GBM) itself to large molecules; this change was focal and was found in those areas where the GBM contained immune complex deposits. Both ferritin and catalase, tracers normally restricted from passing the glomerular filter, were present in the urinary space within minutes of injection. No evidence was obtained for increased glomerular epithelial transport in this disease. Foot process swelling and "close" junction formation was moderate, even in animals with marked degrees of proteinuria. Indirect evidence, therefore, makes an alteration in the slit pore complex likely. In addition, there was immediate and selective concentration of all tracers within deposits, though ferritin was partially excluded from some deposits. This phenomenon may be of significance in the perpetuation of the disease.

Location of T cell and major histocompatibility complex antigens in the human thymus.

A series of monoclonal antibodies were used to study the intrathymic distribution of T cell-specific antigens, Ia antigens, and beta 2-microglobulin in frozen sections of human thymus by immunofluorescence and immunoperoxidase techniques. Most of the cortical thymocytes reacted with anti-T4, anti-T5, anti-T6, anti-T8, and anti-T10 antibodies, thus indicating coexpression of multiple antigens on cortical lymphocytes. The staining of cells in the medulla was most satisfactorily judged in sections stained with the immunoperoxidase technique. Many medullary cells reacted with anti-T4--and a smaller fraction with anti-T5, anti-T6, anti-T8, and anti-T10 antibodies. In addition, T1 and T3 antibodies, which react with all peripheral T cells, stained a majority of medullary cells. The medullary cells were also more intensely stained with antibodies directed against beta 2-microglobulin than the majority of cortical cells. Hence, the staining profile of medulla approximates the staining pattern of peripheral T cells, with large numbers of cells bearing T1+, T3+, and T4+ antigens (helper/inducer cells) and a small number of cells bearing T1+, T3+, and T5+/T8+ antigens (suppressor/cytotoxic cells). This supports the conclusion that mature cells present in the medulla are derived from immature cells in the cortex. However, a small number of cells scattered throughout the cortex stained with T1 and T3 antibodies, which suggests that maturation of thymocytes can also occur in the cortex. Antibody directed against Ia antigens resulted in a characteristic patchy pattern of staining in the cortex and in diffuse staining in the medulla, which was interpreted as resulting from staining of epithelial reticulum. The majority of thymocytes did not stain. The staining pattern suggests a close relationship between epithelial cells and thymocytes.

Distribution of T cell subsets in human lymph nodes.

A series of T cell-specific monoclonal antibodies was used to determine the location of T lymphocyte subpopulations in frozen sections of human lymph nodes by means of an immunoperoxidase technique. The majority of cells in the paracortical regions were reactive with anti-T1 and anti-T3 antibodies, which define all mature peripheral T cells. In contrast, the majority of cells within primary follicles were unreactive with anti-T1 and anti-T3 antibodies, but were reactive with anti-Ia and anti-IgM antibodies. In addition, a substantial number of T1+, T3+ cells were found in the germinal centers of secondary follicles on the capsular side. The vast majority of T1+, T3+ cells in the paracortex and the follicles were reactive with anti-T4 antibody, which defines inducer/helper T cells. Only a minority of cells in these areas were reactive with anti-T5 and anti-T8 antibodies, which define cytotoxic/suppressor cells. No lymphocytes were stained with anti-T6 antibody, which reacts with a majority of thymocytes but not with peripheral T cells. Scattered cells in the paracortex showed staining for Ia antigen in an irregular dendritic pattern. The findings demonstrate that the major T cell population found within human lymph node bears the mature T1+, T3+, T4+ phenotype characteristic of inducer T cells. Moreover, the location of this population indicates that they play a role in the induction of B cell differentiation in vivo.

Stages of B cell differentiation in human lymphoid tissue.

Monoclonal antibodies reactive with B cell-specific differentiation and other antigens were used to investigate stages of B cell maturation in human lymphoid tissue, using an immunoperoxidase technique on frozen tissue sections. Lymphoid follicles, which represent the major anatomic compartment of B cells, demonstrated cellular antigenic expressions that appear to reflect differentiation of B cells. The majority of cells in the primary follicles and the mantle zones of secondary follicles expressed surface antigens similar to those of circulating B cells, namely IgM, IgD, Ia, B1, and B2. In contrast, the germinal center cells of secondary follicles stained for IgM, IgG, B1, B2, and Ia antigens, but not for IgD, and furthermore, acquired the T10 antigen. The germinal centers stained much more intensely than mantle zones with anti-B2, whereas no such striking difference in the staining intensity was observed with anti B1. Plasma cells, which represent the end stage of B cell differentiation, showed intense cytoplasmic staining with the anti-T10 antibody. The results indicate that the generation of germinal center cells in primary lymphoid follicles involves phenotype changes that correspond largely to those previously observed after both antigenic and mitogenic activation of B lymphocytes.

Antibodies to intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 prevent crescent formation in rat autoimmune glomerulonephritis.

In patients with glomerulonephritis widespread crescents are associated with a poor prognosis. Crescent formation appears to depend on the migration of mononuclear cells into Bowman's space, and therefore the interaction between leukocytes and glomerular endothelium may be a critical event in the genesis of crescents. We performed the present study to determine the effects of mouse monoclonal antibodies to the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 1 (LFA-1) in a model of crescentic glomerulonephritis in Wistar-Kyoto rats, induced by immunization with bovine glomerular basement membrane (GBM). By 10-14 d after immunization, the rats had developed circulating anti-GBM antibodies, reactive with the alpha 3 chain of type IV collagen (the Goodpasture antigen), accompanied by proteinuria, accumulation of rat immunoglobulin (Ig)G in the GBM, increased expression of ICAM-1 by glomerular endothelial cells, infiltration of glomerular tufts with LFA-1+ T cells and monocyte/macrophages, and early crescents. At 5 wk all rats had diffuse fibrocellular crescents, glomerular sclerosis, and tubulointerstitial damage. All rats developed severe renal insufficiency and died by 5 or 6 wk. The administration of monoclonal antibodies to rat ICAM-1 and LFA-1 markedly decreased the severity of the renal disease. In a group of rats injected three times a week with the monoclonal antibodies, from 2 d before immunization with GBM to day 14, glomerular abnormalities and proteinuria were virtually absent at day 14; even at 5 wk glomerular disease was quite mild, with only slight crescent formation and with only a mild decrease in renal function. When treatment was continued until 5 wk, the beneficial effects were even more marked, with virtual absence of crescents and with preservation of normal renal function. In a group of rats in which treatment was initiated on day 14, shortly after the appearance of glomerular abnormalities, progression of the disease was appreciably retarded, and the decrease in renal function was inhibited. The kidneys of rats treated from days -2 to 14 with antibodies to ICAM-1 and LFA-1 showed bright linear staining for rat IgG along the GBM, which did not differ in intensity from that seen in untreated rats. Furthermore, the titers of anti-GBM antibodies at 2 wk in treated rats were not lower than that seen in most of the untreated rats. There was, however, moderate reduction of anti-GBM antibodies at 5 wk in the treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor.

The pathogenesis of Heymann nephritis, a rat model of human membranous glomerulonephritis, depends on the interaction of autoantibodies with a renal glycoprotein (GP330) on glomerular podocytes. Partial complementary DNAs coding for GP330 were isolated and sequenced. The deduced amino acid sequence from 4.3 kilobases of complementary DNA contains the sequences identical to two peptides derived from the isolated glycoprotein. The deduced amino acid sequence of this protein contains regions with homology to the human low density lipoprotein (LDL) receptor, an indication that GP330 and the LDL receptor may be members of the same gene family. Autoantibodies from the kidneys of rats with Heymann nephritis reacted with a nonglycosylated segment of GP330 that contains cysteine-rich 40-amino acid repeats, which are also features of the LDL receptor. GP330 is also similar in some regions to the mouse epidermal growth factor precursor.

Binding of the low density lipoprotein receptor-associated protein (RAP) to thyroglobulin (Tg): putative role of RAP in the Tg secretory pathway.

The 39-44 kDa protein known as the receptor-associated protein binds to members of the low density lipoprotein receptor family and is found within cells that express these receptors. The receptor-associated protein has been shown to prevent premature binding of ligands to the receptors in the endoplasmic reticulum and to promote proper folding and transport of the receptors in the secretory pathway. In the thyroid, megalin (a low-density lipoprotein receptor family member) serves as an endocytic receptor for thyroglobulin. Here we present evidence that the receptor-associated protein can bind to thyroglobulin, which suggests a novel function of the receptor-associated protein, namely binding of certain megalin ligands possibly during the biosynthetic pathway. In solid-phase assays thyroglobulin was shown to bind to the receptor-associated protein with moderately high affinity (mean between K(d) and K(i) = 39.8 nM), in a calcium-dependent and saturable manner. The receptor-associated protein also bound to a native carboxyl-terminal 230-kDa thyroglobulin polypeptide, which markedly reduced binding of intact thyroglobulin to the receptor associated protein, indicating that the receptor-associated protein binding sites of thyroglobulin are located in the carboxyl-terminal portion of the molecule. In addition to thyroglobulin, the receptor-associated protein specifically bound to another megalin ligand, namely lipoprotein lipase. Because lipoprotein lipase markedly reduced receptor-associated protein binding to thyroglobulin, we concluded that the receptor-associated protein uses the same binding site/s to bind to thyroglobulin and lipoprotein lipase. Evidence of thyroglobulin binding to the receptor-associated protein was also obtained in vivo and in cultured thyroid cells. Thus, anti-receptor-associated protein antibodies precipitated intact thyroglobulin from extracts prepared from rat thyroids and cultured thyroid cells (FRTL-5 cells). Chase experiments after inhibition of protein synthesis in FRTL-5 cells showed that thyroglobulin interacts with the receptor-associated protein shortly after the beginning of thyroglobulin biosynthesis.

The syndrome of lung hemorrhage and nephritis is usually an ANCA-associated condition.

BACKGROUND: In the absence of evidence of arteritis or Wegener's granulomatosis, the syndrome of lung hemorrhage and nephritis has been commonly associated with anti-glomerular basement membrane (GBM) antibodies. However, it has been increasingly recognized that many cases are associated with antineutrophil cytoplasmic antibodies (ANCAs). OBJECTIVE: To review available clinical and pathologic findings to determine the diseases accounting for lung hemorrhage and nephritis. METHODS: We studied the records of 750 patients from whom serum samples were sent to our laboratory for anti-GBM antibody assays between 1981 and 1993 and found 88 patients with evidence of lung hemorrhage and nephritis. Serum samples were retested, using current methods, for anti-GBM antibodies (against noncollagenous 1 domain of the alpha 3 chain of type IV collagen) and for antibodies to proteinase 3 and myeloperoxidase--the two types of ANCA of diagnostic value. RESULTS: Of 88 patients with evidence of lung hemorrhage and nephritis, 48 had ANCAs, six had anti-GBM antibodies, and seven had both. In 48 patients with ANCAs, the pathologic findings that accounted for the pulmonary renal syndrome were pauci-immune necrotizing and crescentic glomerulonephritis and pulmonary capillaritis. Only eight had convincing evidence (during life) of Wegener's granulomatosis and only one other had documented arteritis. In 27 patients without ANCAs or anti-GBM antibodies, a variety of unrelated renal and pulmonary diseases were found. CONCLUSIONS: The largest group of patients who present with the syndrome of lung hemorrhage and nephritis have ANCAs and not anti-GMB antibodies. Appropriate tests for antibodies to proteinase 3, antibodies to myeloperoxidase, and anti-GBM antibodies provide reliable guides for making a diagnosis in patients with this pulmonary renal syndrome.

Location of gp330/alpha 2-m receptor-associated protein (alpha 2-MRAP) and its binding sites in kidney: distribution of endogenous alpha 2-MRAP is modified by tissue processing.

The alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP) is a 39 to 44 kDa protein that copurifies with the alpha 2-macroglobulin receptor (alpha 2-MR/LRP) and also with gp330, a highly glycosylated protein located within kidney proximal tubules and glomerular podocytes. Both gp330 and the alpha 2-macroglobulin receptor are members of the low density lipoprotein receptor family but the physiological ligands for gp330 are unknown. In order to understand potential functions of the alpha 2-MRAP, specific anti-alpha 2-MRAP antibodies were used for immunocytochemical studies on paraformaldehyde lysine periodate (PLP)-fixed rat kidneys and on snap-frozen/acetone-fixed tissue. Conflicting results were obtained. After PLP fixation, alpha 2-MRAP was detected almost exclusively in rough endoplasmic reticulum (RER) cisternae; cell surface staining was virtually absent. In snap-frozen tissue, intense staining of the proximal tubule brush border was found, with little or no cytoplasmic staining. A series of experiments showed that during incubation of snap-frozen tissues, endogenous alpha 2-MRAP is released in soluble form from its intracellular location (i.e., the RER) and binds to gp330 on the brush border of proximal tubules. The location of binding sites for alpha 2-MRAP in rat kidney was also examined, using an alpha 2-MRAP-IgG fusion protein. In both snap-frozen and PLP-fixed tissues, this probe bound exclusively to brush borders, and not to intracellular sites. Our results demonstrate: a) that in renal proximal tubule cells, alpha 2-MRAP is located predominantly in the RER, b) that alpha 2-MRAP-binding sites are present on gp330, which is on the proximal tubule brush border, and c) that the apparent brush border localization of alpha 2-MRAP detected in snap-frozen sections is due to an artifactual redistribution of endogenous alpha 2-MRAP that occurs during tissue processing.