LOCALIZATION OF IMMUNOLOGICAL COMPLEXES FIXING β1C (C3) IN GERMINAL CENTERS OF LYMPH NODES

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the β_1C component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit β_1C, IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, β_1C, and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for β_1C, IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the β_1C component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.     

AUTOLOGOUS IMMUNE COMPLEX NEPHRITIS INDUCED WITH RENAL TUBULAR ANTIGEN : I. IDENTIFICATION AND ISOLATION OF THE PATHOGENETIC ANTIGEN

The nephritogenic antigen, responsible for the immunogenic stimulus in experimental allergic glomerulonephritis induced with tubular antigen, has been identified as a renal tubular epithelial (RTE)-specific antigen and has been isolated in a relatively purified form. This antigen, RTE-α₅, is a distinct and antigenically specific lipoprotein of high density which is derived primarily from the brush border of proximal convoluted tubular epthelium of the rat kidney. It has been suggested that this molecule may be a plasma membrane subunit. Immunization of rats with as little as 3 µg N of RTE-α₅ in complete Freund's adjuvant has effectively induced this form of membranous glomerulonephritis. RTE-α₅ is not a constituent of normal rat glomeruli; however, with the onset of autologous immune complex nephritis it is deposited in a granular fashion along glomerular capillary walls indistinguishable from the deposits of γ-globulin and complement. The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.     

Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance

Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α₃ or β₁ integrin, or the α₃β₁ ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β₁ integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.     

THE GLOMERULAR MESANGIUM : III. ACUTE IMMUNE MESANGIAL INJURY: A NEW MODEL OF GLOMERULONEPHRITIS

A mechanism of immune glomerular injury is described based on the fixation of antibody (Ab) to an antigen (Ag) that has localized in the glomerular mesangium. Rabbits were given, intravenously (i.v.), aggregated human IgG (AHIgG) or albumin (AHSA) and 10 h later, when the Ag by immunofluorescent microscopy was present in the mesangium, a kidney was removed and transplanted into a normal rabbit. The recipient then received, i.v., rabbit anti-HIgG or anti-HSA. Within minutes of Ab infusion, glomeruli of the donor kidney had polymorphonuclear (PMN) infiltration that over the next few hours became marked and was associated with glomerular cell swelling. At 24 h a decrease in PMN's and early mesangial proliferation was seen. By 3 days there was marked mesangial hypercellularity and increased mesangial matrix. Within minutes after Ab administration rabbit IgG, C3, and fibrin were seen in the glomerular mesangium. There was a fall in complement titer by 1 min after Ab infusion that was due to complement consumption by the donor kidney. Complement then returned to normal levels by 48 h. Significant glomerular injury did not occur (a) in the recipient's own kidney, (b) from Ag administration and transplantation without recipient Ab administration, or (c) from transplantation and Ab administration without prior Ag administration. These studies demonstrated that Ag localized in the glomerular mesangium can react with circulating Ab and complement resulting in severe glomerular injury.     

THE RENAL HANDLING OF HEMOGLOBIN : I. GLOMERULAR FILTRATION

The glomerular filtration of hemoglobin (α₂β₂) was studied under conditions in which its dissociation into αβ dimers was experimentally altered. Rats receiving hemoglobin treated with the sulfhydryl reagent bis(N-maleimidomethyl) ether (BME) showed a much lower renal excretion and prolonged plasma survival as compared with animals injected with untreated hemoglobin. Plasma disappearance was also prolonged in dogs receiving BME hemoglobin. Gel filtration data indicated that under physiological conditions, BME hemoglobin had impaired subunit dissociation. In addition, BME hemoglobin showed a very high oxygen affinity and a decreased rate of auto-oxidation. Glomerular filtration was enhanced under conditions which favor the dissociation of hemoglobin into dimers. Cat hemoglobin, which forms subunits much more extensively than canine hemoglobin, was excreted more readily by the rat kidney. The renal uptake of ⁵⁹Fe hemoglobin injected intra-arterially into rabbits varied inversely with the concentration of the injected dose.     

THE IMMUNOGLOBULINS OF MICE : V. THE METABOLIC (CATABOLIC) PROPERTIES OF FIVE IMMUNOGLOBULIN CLASSES

The metabolic properties of immunoglobulin were investigated by comparing five classes of mouse immunoglobulin. Three forms of 7S immunoglobulin had different rates of catabolism. The fractional rates of catabolism were found to be about 13 per cent per day for 7S γ_2a-globulin; 25 per cent for 7S γ_2b-globulin; and 17 per cent for 7S γ₁-globulin. Catabolism of the three classes of 7S γ-globulin (γ_2a, γ_2b, and γ₁) were prolonged at low serum 7S γ-globulin levels and accelerated at high serum 7S γ-globulin levels. Each of the 7S γ-globulin components was influenced by the serum level of the other mouse 7S γ-globulin components and by exogenously administered human 7S γ-globulin. They were not appreciably altered, however, by the serum level of IgA (γ₁A-, β₂A-globulin). The progressively changing (longer) half-times observed in turnover studies of normal IgG (7S γ-globulin) may be caused by catabolic heterogeneity of normal 7S immunoglobulins which are immunochemically and catabolically related to γ_2a-, γ_2b-, and 7S γ₁-myeloma proteins. These studies indicate that the 7S γ_2a-, 7S γ_2b-, and 7S γ₁-globulins share a common catabolic control mechanism. This mechanism is influenced by the serum level of each of these components, but is independent of the serum level of IgA (γ₁A-globulin) and probably is independent of IgM (γ₁M-globulin). Catabolism of IgA (γ₁A-, β₂A-globulin) and IgM (γ₁M-globulin) was much more rapid than the catabolism of the 7S γ-globulins. The halftimes of the IgA and IgM were approximately 1.2 and 0.5 days respectively. The fractional rate of catabolism of IgA and IgM seemed to be independent of their serum concentration. The rate of catabolism, as well as the rate of synthesis, was shown to play a major role in determining the serum level of each class of immunoglobulin.     

Experimental Membranous Glomerulonephritis in Rats: QUANTITATIVE STUDIES OF GLOMERULAR IMMUNE DEPOSIT FORMATION IN ISOLATED GLOMERULI AND WHOLE ANIMALS

Quantitation of immune deposit formation in glomeruli and correlation with immunohistologic and functional changes has been accomplished only in models of anti-glomerular basement membrane antibody-induced nephritis, or indirectly in immune complex disease by measuring radiolabeled antigen deposition. The kinetics of subepithelial immune deposit formation and the relationship between the quantity of antibody deposited and proteinuria are defined here for the first time in an established model of membranous immune complex nephritis (passive Heymann nephritis) induced by a single intravenous injection of ¹²⁵I-labeled sheep immunoglobulin (Ig)G antibody to rat tubular brush border antigen (Fx1A). Measurement of antibody deposition in glomeruli (GAb) isolated from rats injected with 10 mg of anti-Fx1A demonstrated a mean of 12 μg GAb in 4 h, which increased linearly to 48 μg in 5 d. GAb represented only 20 and 44% of total kidney antibody binding at these times. Proteinuria occurred only after 4-5 d of antibody deposition in rats with total kidney antibody binding exceeding ~200 μg/2 kidneys. Steroid treatment and vasoactive amine blockade did not significantly alter the quantity or localization of immune deposits. It was also demonstrated that isolated rat glomeruli specifically bound nephritogenic quantities of anti-Fx1A in vitro within hours. Analysis of the quantitative aspects of glomerular antibody deposition in vivo and glomerular antibody binding in vitro provides additional evidence that subepithelial immune deposits in passive Heymann nephritis may form in situ by reaction of free antibody with antigenic constitutents of the normal rat glomerulus. The observed kinetics of deposit formation differ markedly from those in anti-glomerular basement membrane disease and suggest a role for factors in addition to antigen-antibody interaction in determining this unique pattern of glomerular immune deposit formation.     

Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension.

Two groups of adult male Munich-Wistar rats and a third group of nondiabetic age-matched and weight-matched normal control rats underwent micropuncture study 1 mo, and morphologic studies 14 mo, after induction of streptozotocin diabetes or sham treatment. All animals were fed standard rat chow. Diabetic rats received daily ultralente insulin to maintain stable moderate hyperglycemia (approximately 350 mg/dl). In addition, one group of diabetic rats was treated with the angiotensin I converting enzyme inhibitor, enalapril, 15 mg/liter of drinking water. Average kidney weight, whole kidney and single-nephron glomerular filtration rate, and glomerular plasma flow rate were elevated to similar values in both groups of diabetic rats, relative to normal control rats. Non-enalapril-treated diabetic rats exhibited significant elevations in mean glomerular capillary hydraulic pressure and transcapillary hydraulic pressure gradient, compared with the other groups studied, and only this group eventually developed marked and progressive albuminuria. Likewise, histological examination of the kidneys at 14 mo disclosed a high incidence of glomerular structural abnormalities only in non-enalapril-treated diabetic rats. These findings indicate that prevention of glomerular capillary hypertension in rats with diabetes mellitus effectively protects against the subsequent development of glomerular structural injury and proteinuria. This protection is afforded despite pronounced hyperglycemia and elevated levels of glucosylated hemoglobin, further supporting our view that hemodynamic rather than metabolic factors predominate in the pathogenesis of diabetic glomerulopathy.     

ELECTRON MICROSCOPIC STUDIES OF HUMAN GLOMERULONEPHRITIS WITH FERRITIN-CONJUGATED ANTIBODY : LOCALIZATION OF ANTIGEN-ANTIBODY COMPLEXES IN GLOMERULAR STRUCTURES OF PATIENTS WITH ACUTE GLOMERULONEPHRITIS

1. Kidney biopsies from 4 cases of severe acute glomerulonephritis were obtained 11 to 25 days after the onset of clinical manifestations of the disease. These tissues were treated with ferritin-conjugated antibodies to 7S γ-globulin, β_1C, and Type 12 streptococcal products. Adjacent pieces of the biopsied material were treated with control ferritin-labeled antisera or with ferritin alone. As further controls, normal renal tissue and renal tissue from patients with other kidney diseases were treated with the same antisera. The 3 antisera to 7S γ-globulin, β_1C and Type 12 streptococcus were specifically bound in electron-opaque foreign material in the following renal areas: (a) the lumen of glomerular capillaries; (b) medullary arteriolar walls (2 cases); (c) pinocytic vacuoles and absorption droplets of endothelial or mesangial cells; (d) canals between proliferating mesangial or endothelial cells which connect the capillary lumen with the deep mesangial region or with the endothelial side of the basement membrane; (e) basement membrane proper; (f) subendothelial and certain subepithelial deposits; and (g) Bowman's space. 2. None of the 3 ferritin-conjugated antisera listed above were bound to the nuclei of glomerular cells or to portions of the cytoplasm other than those specified. 3. Ferritin-conjugated antisera to pneumococcus Type II and vaccinia virus and ferritin alone were not bound to any structures in the glomerular tissue. 4. None of the ferritin-conjugated antisera bound to normal renal tissue or to kidney tissue from other renal disease. 5. The data obtained are compatible with the following working hypothesis: Antigen-antibody aggregates of Type 12 streptococcal products, γ-globulin, and complement are present in the circulating blood of patients with severe acute glomerulonephritis. Large amounts of the complexes are caught in the filtering system of the glomeruli. The inflammatory reactions seen in the glomerular structures result from the presence of the immune complexes and of the polymorphonuclear leukocytes which conjointly may be responsible for the disease.     

β2-microglobulin–deficient Mice Are Resistant to Bullous Pemphigoid

Recent understanding of the mechanism of immunoglobulin G (IgG) catabolism has yielded new insight into antibody-mediated diseases. We proposed that β₂-microglobulin (β₂m)–deficient mice have been protected from systemic lupus erythematosis (SLE)–like syndromes because they lack the β₂m-associated IgG protection receptor (FcRn) and therefore catabolize IgG, including pathogenic IgG autoantibodies, considerably more rapidly than normal mice. Such an hypothesis would predict that β₂m-deficient mice would also be resistant to experimental bullous pemphigoid, a disease with a pathogenesis thought to be much simpler than SLE, being the result of antibody directed toward a pathogenic epitope on the epidermal hemidesmosome that anchors basal keratinocytes to the basement membrane. To test this hypothesis, we administered pathogenic rabbit antibody directed toward the hemidesmosome to β₂m-deficient mice and to normal control mice, both intraperitoneally and intradermally, and assessed the mice clinically, histologically, and immunologically for manifestations of skin disease. We found that the β₂m-deficient mice were protected when the antibody was given intraperitoneally whereas intradermal administration resulted in blisters only slightly less severe than those seen in normal mice. These data would indicate that autoantibody-mediated inflammation might be prevented or controlled by appropriate modulation of FcRn function.     

CHARACTERISTICS OF IMMUNOLOGICAL MEMORY IN MICE : I. SEPARATE EARLY GENERATION OF CELLS MEDIATING IGM AND IGG MEMORY TO SHEEP ERYTHROCYTES

The kinetics of the generation of primed IgM and IgG antibody-forming cell precursors, and of helper T-cell populations, were analyzed in mice whose primary responses to high and low doses of SRBC were arrested at intervals by the immunosuppressive agents cyclophosphamide monohydrate and specific antibody. The extent to which immunological memory was established in these animals before blockade of the primary response was assessed by the hemolytic plaque assay following challenge 12 wk after priming. The presence of IgG B-memory cells and T-memory cells in suppressed mice was further investigated by the transfer into these animals of syngeneic SRBC-stimulated thymocytes or anti-θ-treated spleen cells. It was found that the progenitors of secondary IgM-synthesizing cells were primed almost immediately after injection of antigen, and that early blockade of the primary response resulted in a raised IgM response after challenge. On the other hand, priming for a secondary IgG response took at least 4 days, and was dose-dependent, although helper T populations for a secondary IgG response appeared 3 days after antigen injection. It appeared that both IgM and IgG memory cells may be considered as Y cells in terms of the X-Y-Z scheme of lymphocyte activation, but that the two populations are generated at different times after exposure to antigen. The size of either Y-cell population at any given time is dependent upon the amount of antigen available to provoke differentiation to antibody-forming Z cells, and the IgM Y-cell population in particular is likely to be depleted during the course of a normal 1° response. When IgM Y cells were maintained for long periods as a result of immunosuppression, their secondary antibody response was independent of the primed T cells necessary for a secondary IgG response.     

the relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. III. Expression of a single predominant isotype on primed and unprimed B cells

We determined whether primed and unprimed B cells in the spleen of (BALB/c × C57BL/Ka)F(1) mice contain subpopulations that express a predominant surface Ig isotype. Spleen cells were stained for surface isotypes and sorted on the fluorescence-activated cell sorter (FACS) in order to obtain B cells bearing predominantly IgM (μp cells), IgD (δp cells), or IgG (γp cells). Each population was assayed for its capacity to restore the adoptive primary and secondary anti-bovine serum albumin (BSA) antibody response in irradiated syngeneic recipients. In addition, the adoptive response restored by isotype-predominant cells was compared to that restored by isotype- positive cells (B cells bearing a given surface isotype alone or in combination with others). The experimental results show that μp cells restore the adoptive primary and secondary IgM and IgG responses to BSA, and γP cells restore only the primary and secondary IgG response. Δp Cells restored the adoptive secondary IgG response, but failed to restore the adoptive primary response at the cell doses tested. ΓP Cells but not δp cells suppressed the IgM response of the μ(+) and δ(+) cells. The contribution of isotype-predominant cells to both the adoptive primary and secondary anti-BSA response was smaller than that of B cells bearing a combination of surface isotypes. Differences in the Ig isotype pattern expressed on the surface of primed and unprimed B cells are discussed.     

Immunoglobulin isotypes in the circulating immune complexes of diabetic rats with and without proteinuria: a chronological study

Circulating immune complexes (CIC) have been postulated to contribute to the development of the secondary complications of diabetes mellitus. Therefore studies were performed to determine whether CIC are the cause of the consequence of the development of diabetic nephropathy. This was done by comparing the occurrence and concentration of CIC containing the different isotypes of immunoglobulins in control rats to those detected in streptozotocin-induced (Sz) diabetic rats that developed albuminuria (Group I) and that did not develop albuminuria (Group II). Only CIC containing IgM, IgG2b and IgG2c were detected in diabetic rats. By staging Group I albuminuric diabetic rats to a clinical reference point of albuminuria, there was no correlation between the occurrence or concentration of CIC containing any isotype of immunoglobulin and the onset of albuminuria. In all Group I albuminuric diabetic rats, the occurrences of all CIC were variable and their concentrations fluctuated during the development and early progression of nephropathy. However, after this group of diabetic rats progressed to overt nephropathy (marked by albuminuria and IgG proteinuria), CIC could be demonstrated in 100% of the animals. In diabetic rats that did not develop albuminuria (Group II), CIC containing IgG2b occurred earlier and more often than in Group I albuminuric rats. Similarly, the subclass IgG2c was detected in the CIC of Group II non-albuminuric rats more frequently and in higher concentrations than in Group I albuminuric rats. CIC containing IgM were detected in all 3 animal groups, however, in higher concentrations and occurrences in Group II non-albuminuric rats as compared to control and Group I albuminuric rats. The consistent elevation in CIC after the development of diabetic nephropathy, suggests that the CIC containing any immunoglobulin isotype either result from diabetic kidney, or from other deteriorating conditions associated with the diabetic state. The data also suggests that CIC are not involved in the onset or progression of diabetic nephropathy regardless of the isotypes of immunoglobulins contained within the CIC. However, there is an isotypic restriction in the immunoglobulins detected in the CIC of diabetic rats (IgM, IgG2b and IgG2c) that may signal some involvement of the immune system in the development of diabetic nephropathy.     

THE RELATIONSHIP BETWEEN β2-MICROGLOBULIN AND IMMUNOGLOBULIN IN CULTURED HUMAN LYMPHOID CELL LINES

β₂-microglobulin was detected on the cell surface and in the medium of human lymphoid cells established in long-term culture. The secretion of β₂-microglobulin was relatively uniform when different cell lines were compared, whereas IgG production varied widely. κ- and µ-membrane antigens were modulated by specific antibody; β₂-microglobulin was not modulated. Anti-κ and anti-µ antisera had no effect on the expression of membrane β₂-microglobulin, nor had anti-β₂-microglobulin antiserum any effect on the expression of κ- and µ-membrane antigens.     

IMMUNE RESPONSES IN VITRO : V. SUPPRESSION OFγM, γG,ANDγA PLAQUE-FORMING CELL RESPONSES IN CULTURES OF PRIMED MOUSE SPLEEN CELLS BY CLASS-SPECIFIC ANTIBODY TO MOUSE IMMUNOGLOBULINS

Suppression of Ig class-specific PFC responses by class-specific antibody to mouse immunoglobulin was studied in cultures of spleen cells from immunized mice. In contrast to cultures from normal mice where anti-µ suppressed responses in all Ig classes, anti-µ had progressively less suppressive effect on γ₁ and γ₂ responses in cultures from immunized mice with time after immunization. This was most pronounced at 10 days after immunization when anti-µ suppressed γM and γA responses, but had no or slight effect on γ₁ or γ₂ responses which were still suppressed with anti-γ₁ and anti-γ₂. These changes in precursor cell susceptibility to anti-µ were antigen specific.     

Direct Radioimmunoassay of Nuclear 3,5,3′ Triiodothyronine in Rat Anterior Pituitary

Previous tracer studies have suggested that 5′-monodeiodination of l-thyroxine (T₄) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3′ l-triiodothyronine (T₃) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T₃ in individual anterior pituitaries was developed. Animals received [¹²⁵I]T₃ 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T₃ ratio and comparison of expected with measured T₃. T₃ was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T₃ was 0.18 ± 0.06 pg/μg DNA which was 0.13 pg/μg DNA greater than expected from the serum T₃ concentration and the pituitary nuclear:serum [¹²⁵I]T₃ ratio. In 10 hypothyroid rats given a single bolus of 400 ng T₃/100 g body wt., the nuclear T₃ by radioimmunoassay was 1.0 ± 0.06 pg/μg DNA, whereas that expected from the T₃ specific activity calculations was 0.85 pg/μg DNA (P < 0.025). Serum T₄ concentrations in these rats were < 0.25 μg/dl but the nuclear T₃ derived from as little as 0.2 μg/dl T₄ could explain a large portion of these small discrepancies between observed and measured nuclear T₃. In 29 normal rats, anterior pituitary nuclear T₃ was 0.63±0.04 pg/μg DNA, whereas that expected from the serum T₃ concentration (55±2 ng/dl) was 0.23±0.02 pg/μg DNA (P < 0.001). Total pituitary T₃ based on this measurement was 92±6 pg. Because the maximal nuclear binding capacity for T₃ in rat anterior pituitary is 0.77 pg/μg DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T₄ and T₃ to achieve normal nuclear T₃ saturation in anterior pituitary is in marked contrast to the situation in liver, kidney, and heart muscle which appear to require only a normal serum T₃. As a consequence, the anterior pituitary can monitor both serum T₄ and T₃ and respond appropriately to changes in their concentrations.     

Lymphotoxin-α (LTα) Supports Development of Splenic Follicular Structure That Is Required for IgG Responses

LTα-deficient (LTα^−/−) mice show altered splenic microarchitecture. This includes loss of normal B cell–T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of ability to form germinal centers (GC). LTα^−/− mice immunized with sheep red blood cells (SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LTα expression by lymphocytes cooperating in the antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LTα^−/− mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LTα^−/− mice, follicle structure and a strong IgG response were retained. These data indicate that LTα-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTα^−/− mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of LTα could alter the structure and function of spleen follicles, reciprocal bone marrow (BM) transplantation was performed. Six weeks after reconstitution of LTα^−/− mice with wild-type BM, spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with LTα^−/− BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather, expression of LTα supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG response.Lymphotoxin-α (LTα)¹ shares structural features with the related cytokine, TNFα. Both LTα and TNFα exist in solution as homotrimeric proteins. In these forms, they share biological activities by virtue of their similar binding to the two defined TNF receptors, TNFR-I, and TNFR-II. Signaling via these two receptors modulates a wide variety of immune and inflammatory responses (1, 2). LTα also exists in a heteromeric form with the type II membrane protein LTβ, which in its most prevalent form on the membrane has the stoichiometry LTα₁LTβ₂. The LTα₁LTβ₂ heteromer has no measurable affinity for TNFR-I or TNFR-II, but does interact with high affinity with the TNFR-related protein (also designated the LTβ receptor, or LTβR) (3–5).LTα^−/− mice are born with defective development of LN and Peyer''s patches (PP) (6). In LTα^−/− mice, spleen structure is also disturbed, with small white pulp follicles that fail to segregate T cell and B cell zones, and fail to generate clusters of FDC or GC (6–8). Proper spleen microarchitecture, including the presence of primary and secondary lymphoid follicles that contain FDC, is thought to be required for all features of a mature T cell–dependent B cell response, including Ig class switching, affinity maturation, and development of antibody secreting cells (9–11). Consequently, it was striking that LTα^−/− mice were able to generate high affinity anti-NP IgG antibody after immunization with high doses of the T cell–dependent antigen 4-hydroxy3-nitrophenyl-ovalbumin (NP-OVA) adsorbed to alum (8). In contrast, there was impaired production of high affinity anti-NP IgG when LTα^−/− mice were immunized with low doses of NP-OVA absorbed to alum. Banks et al. (12) using an independently derived LTα^−/− mouse strain, have shown impaired IgG responses in LTα^−/− mice after subcutaneous immunization with KLH absorbed to alum or immunization with viral antigens. Further studies examining mice deficient in both TNFα and LTα demonstrated variable IgG responses, with deficient IgG responses after intraperitoneal immunization with SRBC but retained responses against vesicular stomatitis virus after an infectious challenge (13). Recently prepared TNFα^−/− mice (14) are reported also to have an impaired IgG anti-SRBC response but a strong response to DNP-KLH adsorbed to alum. Thus, the role of LTα in supporting an effective IgG response to SRBC remains incompletely defined.LTα^−/− mice manifest a complex phenotype that includes an absence of LTα expression and also established abnormalities of lymphoid tissue development and structure (6–8). The present study was undertaken to investigate the requirements for LTα expression in lymphoid cells and for intact lymphoid tissue structure to support production of an IgG responses to the T cell–dependent antigen SRBC. We report that LTα^−/− mice responded with high levels of IgM but very low levels of IgG after immunization with SRBC in the absence of adjuvant. Experiments in which suspensions of mature spleen cells or of  T cell–depleted bone marrow were transferred to wild-type or LTα^−/− mice demonstrated that certain elements of spleen follicle structure are plastic and are determined by the presence of LTα-expressing cells. These experiments also demonstrated that LTα^−/− B cells and T cells in a structurally intact lymphoid tissue environment are competent to perform Ig isotype switching. In contrast, disturbed lymphoid tissue structure caused by absence of LTα and manifested by absence of clusters of FDC is associated with an inability to form an effective IgG response. Thus, LTα produced by bone marrow (BM)–derived cells establishes a permissive environment for an effective IgG response.     

The glomerular mesangium: I. Kinetic studies of macromolecular uptake in normal and nephrotic rats

This study was designed to define quantitatively the function of the rat glomerular mesangium in the uptake and processing of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, AHIgG-(125)I), to relate this function to that of the general reticuloendothelial system, and to examine the effects of increased glomerular permeability to protein on the mesangial cell system.Mesangial localization of human IgG as demonstrated by immunofluorescent microscopy showed good correlation with concentrations of AHIgG-(125)I in preparations of isolated glomeruli. In normal rats the concentrations of AHIgG-(125)I in glomeruli were similar to those of lung, liver, and spleen and demonstrated a rapid decrease with increasing time intervals after aggregate administration.In rats given aminonucleoside of puromycin a marked increase in mesangial uptake of aggregates was found while studies of nephrotic lungs, liver, spleen, and blood showed no such differences. Glomerular levels of AHIgG-(125)I in aminonucleoside animals could not be correlated with the quantity of proteinuria.Nephrotic and control animals given unaggregated human IgG showed little glomerular localization by immunofluorescent microscopy; no difference in the concentration of this protein in nephrotic as compared to control glomerular isolates was found.Thus, the mesangium in normal animals functions in a manner analogous to that of the general reticuloendothelial system. In nephrotic rats the mesangial uptake of macromolecules is makedly increased, a finding not observed in other tissues.     

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.     

MicroRNA-29b Inhibits Diabetic Nephropathy in db/db Mice

Inflammation and its consequent fibrosis are two main features of diabetic nephropathy (DN), but target therapy on these processes for DN remains yet ineffective. We report here that miR-29b is a novel therapeutic agent capable of inhibiting progressive renal inflammation and fibrosis in type 2 diabetes in db/db mice. Under diabetic conditions, miR-29b was largely downregulated in response to advanced glycation end (AGE) product, which was associated with upregulation of collagen matrix in mesangial cells via the transforming growth factor-β (TGF-β)/Smad3-dependent mechanism. These pathological changes were reversed by overexpressing miR-29b, but enhanced by knocking-down miR-29b. Similarly, loss of renal miR-29b was associated with progressive diabetic kidney injury, including microalbuminuria, renal fibrosis, and inflammation. Restored renal miR-29b by the ultrasound-based gene therapy was capable of attenuating diabetic kidney disease. Further studies revealed that inhibition of Sp1 expression, TGF-β/Smad3-dependent renal fibrosis, NF-κB–driven renal inflammation, and T-bet/Th1-mediated immune response may be mechanisms associated with miR-29b treatment in db/db mice. In conclusion, miR-29b may play a protective role in diabetic kidney disease and may have therapeutic potential for diabetic kidney complication.