Participation of tyrosine kinase in capping, internalization, and antigen presentation through membrane immunoglobulin in BAL17 B lymphoma cells.

BAL17 cells pulsed with goat anti-IgM or anti-IgD as antigens stimulated a goat IgG specific T cell clone in terms of inositol phosphate production. The antigen-presenting capacity of BAL17 cells was inhibited by pretreatment with the tyrosine kinase inhibitors herbimycin A or genistein. Furthermore, ligand-induced capping and endocytosis of membrane immunoglobulin, monitored at the single cell level, was also blocked by herbimycin A. These results indicate that tyrosine phosphorylation plays an important role in receptor-mediated antigen presentation by B cells.

Clinical features and course of aortitis syndrome in Japanese women older than 40 years.

The clinical features and course of aortitis syndrome were studied in 11 women older than 40 years of age. The patients were Japanese women, mean age 57 +/- 6 years old, who were followed for 6.9 +/- 3.8 years. Data from 24 young patients were used for comparison. In the older patients, systemic hypertension (73%), calcification of the aorta (73%), left ventricular hypertrophy (92%) and cardiomegaly (82%) were frequent, whereas the erythrocyte sedimentation rate was normal in 5 patients and only slightly accelerated in 6. C-reactive protein was positive in 2. The incidence of cardiac involvement and inflammatory signs was significantly different from findings in the young patients. Aortic regurgitation (AR) (55%) was significantly more frequent and renal artery stenosis was not observed. Other arterial lesions revealed a pattern similar to those seen in the young patients. An irregular luminal surface, kinking and calcification were present in the lesions in the older patients. The survival rate at 5 years was 80%. Five of 6 patients with AR had congestive heart failure, 4 of whom died. One died after a stroke. Thus, aortitis syndrome in older patients has a long course. There is usually an associated AR, renal artery stenosis is rare and other arterial lesions do not change a great deal. The prognosis may be good, but depends on the association of AR.

Role of newly synthesized MHC class II molecules in antigen-specific antigen presentation by B cells.

Using a B lymphoma, A20-HL, bearing IgM receptors for TNP, we have shown that presentation of an Ag taken up through the receptors is highly sensitive, whereas that of an Ag taken up nonspecifically is resistant to inhibition of protein synthesis or protein transport from the endoplasmic reticulum. To analyze the difference, we have examined the effect of protein synthesis inhibition on A20-HL cells in terms of internalization and fragmentation of a specific Ag, TNP-OVA, and distribution of MHC class II molecules. Inhibition of protein synthesis in A20-HL cells with emetine, an irreversible protein synthesis inhibitor, did not decrease the surface expression of anti-TNP receptors, or the kinetics of internalization of 125I-TNP-OVA. To detect fragmentation of TNP-OVA, A20-HL cells were incubated at 37 degrees C in the presence of 125I-TNP-OVA, and the cell lysate was analyzed in SDS-PAGE. The number of detectable fragments increased with the incubation period, and inhibition of protein synthesis did not change the electrophoretic pattern. Expression of MHC class II molecules on the surface of A20-HL cells was not affected by inhibition of protein synthesis. However, intracellular MHC class II molecules markedly decreased in amount in the emetine-treated cells. Thus, presentation of an Ag taken up through Ag receptors seems to be dependent on intracellular MHC class II molecules, whereas that of an Ag taken up nonspecifically does not, suggesting that the Ag-processing pathway in B cells for a specific Ag is different from that for a nonspecific one, at least partly.

Differential sensitivity to antigenic competition in antigen-specific and -nonspecific antigen presentation by B cells.

We have previously shown that a specific Ag presentation by B cells is different from a nonspecific one in the sensitivity to protein synthesis inhibition. In the present study we have compared the sensitivity of these Ag presentations to antigenic competition. A20-HL cells expressing TNP-specific IgM were pulsed with anti-mouse IgM goat IgG (aMGG) or trinitrophenylated goat IgG (TNP-NGG) as an Ag internalized through Ag receptor or NGG as an Ag internalized by fluid-phase pinocytosis. The pulsed cells induced IL-2 production by NGG-specific cloned T cells. The presence of dog IgG during pulsing A20-HL cells severely inhibited the presentation of NGG but not of aMGG or TNP-NGG. The presence did not decrease the internalization of 125I-NGG into A20-HL cells, suggesting that the inhibition was localized into the complex formation of antigenic peptides with MHC class II molecules. Thus, a specific Ag presentation by A20-HL cells is different from a nonspecific one in its sensitivity to antigenic competition.

Analysis of the autoimmune response in lupus mice: the behaviour and lifespan of anti-DNA-secreting B-cell clones.

We present the results of a study of the physical, haematological and serological features of the progress of the SLE-like syndrome in MRL/Mp-lpr/lpr and (NZB x NZW)Fl mice. As part of this study, we have analysed the IEF spectrotypes of anti-ssDNA antibodies in the sera of these mice and shown that the anti-ssDNA response is clonally restricted, as we have previously shown in a mouse chimaera model and in human SLE. Sequential qualitative and quantitative analysis of anti-ssDNA clonotypes has revealed that the lupus mouse anti-ssDNA clones are relatively short lived, having a lifespan of only 6 to 8 weeks, contrasting sharply with the much longer lifespan previously reported for a mouse anti-DNP-secreting clone and the exceptionally long lifespan of most anti-ssDNA-secreting clones of SLE patients. The implications of these observations for our understanding of the regulation of the autoimmune response are discussed.

Needle aspiration biopsy of vulvar endometriosis. A case report.

Vulvar involvement by endometriosis is extremely rare. A patient presented with a vulvar tumor and was diagnosed on needle aspiration biopsy and subsequently on histopathology as having endometriosis of the vulva. The treatment offered was conservative, local excision of the tumor. The patient was well and free of complaints when last seen in the Outpatient Department, at six months of follow-up. Needle aspiration biopsy as a diagnostic tool in vulvar tumors and the histogenesis of the endometriosis are discussed.

The endoparasitoid Cotesia kariyai (Ck) regulates the growth and metabolic efficiency of Pseudaletia separata larvae by venom and Ck polydnavirus.

It was previously demonstrated that parasitization by Cotesia kariyai caused a decrease in weight gain and food consumption in host larvae, resulting in a lower final weight for parasitized hosts. It is predicted that C. kariyai regulates the physiological condition of the host to obtain maximum food under restricted nutritional conditions. Approximate digestibility (AD) was higher following parasitization but the efficiency of conversion of digested food (ECD) of the parasitized hosts was lower. This suggests that resources available to the parasitoid larvae are enhanced in the parasitized hosts. We evaluated the physiological changes caused by injection of calyx fluid (polydnavirus) plus venom (C+V) in nonparasitized hosts. Injection of C+V into the nonparasitized hosts duplicated the effects of parasitism, namely it increased the AD and decreased the ECD. Furthermore, C+V injections elevated trehalose concentrations in nonparasitized host 7 to 10 d after injection (2nd stadium of the parasitoid larva). Protein content also increased on days 9 and 10 after C+V injection. These results suggest that the nutrients that parasitoid larvae require for their growth increase in the hemolymph of the host during the 2nd stadium of the parasitoid larva.

IL-2 production by B cells stimulated with a specific antigen.

The ability of a specific antigen (Ag) to stimulate B cells to produce IL-2 was examined with a murine B lymphoma line, A20-HL, which expressed surface IgM specific for trinitrophenyl (TNP). The culture supernatant of A20-HL cells stimulated with TNP3.9-ovalbumin (-OVA) or anti-IgM goat IgG contained an activity which supported the proliferation of an IL-2-dependent T cell line, CTLL-2. Neither TNP3.9-OVA nor anti-IgM antibody stimulated the parent line, A20.2J, which did not bear TNP-specific sIg, whereas anti-mouse Ig rabbit IgG F(ab)2 did stimulate both A20-HL cells and A20.2J cells. The active material in the culture supernatant was identified as IL-2 based on the experiments in which the activity was inhibited by anti-IL-2 mAb, and IL-2 mRNA was expressed in A20-HL cells stimulated with TNP3.9-OVA or anti-IgM antibody. These results support the conclusion that a specific Ag can stimulate A20-HL cells to produce IL-2. For IL-2 production, TNP receptors on A20-HL cells have to be appropriately cross-linked, inasmuch as either TNP3.9-OVA or TNP6.7-OVA was much more effective than TNP1.2-OVA and TNP22.9-OVA in the induction of IL-2 production by A20-HL cells.

Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factors derived from autoimmune MRL/MpJ-lpr/lpr mice.

A MRL strain bearing the autosomal recessive mutant gene, lpr (lymphoproliferation), spontaneously develops, in addition to a lupus-like syndrome, unique serological and pathological manifestations. Production of high titers of IgG rheumatoid factors (RF) may be related to the formation of extremely large amounts of cryoglobulins and the development of tissue lesions such as necrotizing polyarteritis, arthritis, and glomerulonephritis. To analyze more directly the relationship of IgG RF to the development of cryoglobulins and tissue injuries, we have established four monoclonal IgG RF secreting hybridomas from unimmunized MRL-lpr/lpr mice and determined their pathogenic effects in normal strains of mice. All the monoclonal IgG RF obtained in this study were of the IgG3 subclass and generated cryoglobulins. However, the fact that not only IgG3 Rf monoclonals but also four of five non-RF IgG3 monoclonals were able to form cryoglobulins, which were composed exclusively of each IgG3 monoclonal, indicates that the IgG3 molecule has a unique physicochemical property to self-associate via nonimmunological interaction and the ability to form cryoglobulins. When the in vivo pathogenic activities of these IgG3 RF and non-RF monoclonals were examined, three of IgG3 RF monoclonals with the specificity to IgG2a were able to induce extensive pathologic manifestations including peripheral vasculitis and glomerulonephritis characteristic of patients with cryoglobulinemia. Our results indicate that the IgG3 itself, independently of its specificity, could be a potential source of cryoglobulins and IgG3 RF, combined with its activity of cryoglobulin formation, may play a significant role in the development of glomerulonephritis and cutaneous vascular lesions of ears and foot pads observed frequently in aged MRL-lpr/lpr mice.

Vulvar dystrophy: a clinical follow-up.

The vulvar dystrophy is an enigma and one of the most controversial topic as for the pathogenesis, clinical outcome and particularly the malignant potentiality. There has been very few systematic studies regarding vulvar dystrophy in Japan. Vulvar dystrophy with dysplasia is said to be a pre-malignant condition of the vulva. So in our institute a prospective follow-up of 5 months to 4 years was obtained in vulvar dystrophic patients after a conservative protocol of treatment. The initial histological diagnosis revealed 17 patients with squamous hyperplasia (9 patients with dysplasia and 8, without dysplasia), lichen sclerosus in 5 and other dermatoses in 1 patient. "Itching" was the most frequent complaint, encountered in 20 (69%) of the study patient. Amongst these patients 4/5 of lichen sclerosus and 16/18 of squamous hyperplasia and other dermatoses had complete symptomatic relief with testosterone and corticosteroid respectively. Morphologically 22/23 (96%) had more than 50% decrease of the total lesion. In squamous hyperplasia with dysplasia after treatment, the re-biopsy revealed improvement of the dysplasia in all the cases with total disappearance in the mild dysplasia group. So far, vulvar carcinoma did not develop in any of the patient followed in our study who received treatment for vulvar dystrophy. So, for vulvar dystrophy, a benign squamous epithelial disorder of the vulva, steroid hormone therapy is indeed a logical and a consistent one.

Immune complexes in Takayasu's arteritis.

We examined sera and Fc receptor-bearing lymphocytes from peripheral blood of patients with Takayasu's arteritis for the purpose of investigating the presence of immune complexes (IC). IC in sera were assayed by solid-phase conglutinin-binding test. Seven of 29 patients exceeded the normal range of circulating IC. IC combined with Fc receptors were estimated by enumerating EA-RFC. EA-RFC of lymphocytes from patients with Takayasu's arteritis were 13.0% and those of normal controls were 29.1%. Low EA-RFC in the patient group increased significantly when lymphocytes were incubated with EA after rising lymphocytes with medium at 37 degrees C. On the contrary, EA-RFC from healthy subjects did not increase after rinsing cells. These findings indicate that IC combined with Fc receptors and hindered EA rosette formation and that rinsing cells with medium at 37 degrees C removed IC from Fc receptors. Comparable results were obtained by a membrane immunofluorescence method using FITC-conjugated anti-human immunoglobulin. In order to confirm that EA rosette formation was really blocked by IC, lymphocytes from a healthy donor were incubated with heat-aggregated human IgG. Incubating cells with IgG aggregates caused reduction of EA-RFC and these lymphocytes restored their ability to form rosettes with EA by rinsing cells with medium at 37 degrees C. In conclusion, we could confirm the presence of IC both in sera and on lymphocyte Fc receptors in some cases of Takayasu's arteritis.

Differences of immunoglobulin secreting cells in bone marrow from those in circulation: relevance to their development.

The surface markers of immunoglobulin secreting cells (ISC) in bone marrow and peripheral blood were analysed. Circulating ISC bear surface Ig and Ia-like antigens. However, these markers were not detectable on ISC in bone marrow. Fc and complement receptors were not present on circulating ISC. The areas of plaques corresponding to Ig secretion by bone marrow cells were always larger than those of peripheral blood cells. Although the majority of ISC were typical plasma cells, plasmacytoid lymphocytes were observed in peripheral blood. These findings seem to indicate that ISC in the peripheral blood are less advanced in their differentiation and maturation pathway from B lymphocytes to plasma cells than those in bone marrow. ISC in mesenteric lymph nodes exhibited nearly the same phenotype as peripheral cells.

Cholera toxin increases intracellular pH in B lymphoma cells and decreases their antigen-presenting ability.

Cholera toxin (CT) can function as a potent adjuvant in the mucosal immune response. However, we have found that treatment of A20-HL murine B lymphoma cells with CT severely inhibits the presentation of ovalbumin (OVA) to cells of the T cell clone 42-6A specific for OVA(323-339)/I-Ad, whereas it does not affect the presentation of OVA(323-339) peptide. CT treatment did not affect the expression of B7-1, B7-2, ICAM-1, LFA-1 or MHC class II on, or the internalization of OVA into A20-HL cells. In CT-treated A20-HL cells, degradation of OVA was decreased, and intracellular pH was raised to a level approximately equivalent to that in CH3NH2-treated cells. Treatment with CH3NH2 is known to raise the pH in endocytic structures and thus inhibits antigen processing. Treatment of A20-HL cells with dibutyryl-cAMP similarly increased intracellular pH. The increase in intracellular pH following CT treatment was inhibited by a cAMP inhibitor, 2',3'-dideoxyadenosine. These results strongly suggest that CT treatment of A20-HL cells inhibits their antigen-presenting cell function by triggering the cAMP cascade, increasing intracellular pH, and reducing the degradation of OVA.

IgG rheumatoid factors and cryoglobulins in mice bearing the mutant gene lpr (lymphoproliferation).

The autosomal recessive mutant gene, lpr (lymphoproliferation), produces a massive proliferation of T cells in autoimmune MRL mice. Since the MRL strain bearing the lpr mutation is the only strain that spontaneously develops high titers of rheumatoid factors (RF), we have investigated whether non-autoimmune mice (C57BL/6, C3H/HeJ and AKR) bearing the lpr gene could produce RF. Serum levels of RF activity were assessed by a new radioimmunoassay, in which serum samples pretreated with acetate buffer were incubated with 125I-mouse IgG and precipitated with 7% polyethylene glycol. The majority of serum from 4- to 6-month-old non-autoimmune strains of mice bearing the lpr gene exhibited significant RF activity, as did MRL-lpr/lpr mice. Sucrose density-gradient analysis has revealed that all the IgG RF activity was present in a form of immune complexes, sedimenting in the intermediate position (7-19S) and fully dissociable into 7S IgG under an acid condition. This indicates that the production of IgG RF does not require a particular background genome of the MRL strain. In addition, mice bearing the lpr mutation developed extremely large amounts of cryoglobulins, which paralleled the production of RF. Analysis of components of their cryoglobulins revealed a marked enrichment of IgG3 subclass as compared to other IgG subclasses and IgM. These results suggest that IgG3-containing immune complexes represent the major source of cryoglobulins occurring in mice bearing the lpr gene.

Tyrosine protein kinase is involved in anti-IgM-mediated signaling in BAL17 B lymphoma cells.

BAL17 B lymphoma cells, representing mature B lymphocytes, were used to analyze the role of tyrosine kinase in B cell activation. Anti-IgM-induced tyrosine phosphorylation was inhibited by preincubation of cells with tyrosine kinase inhibitor herbimycin A. Enzymatic activity of lyn protein was also inhibited by this drug, accompanied by down-regulation of p53lyn and p56lyn. However, a protein kinase C-mediated event was intact in the herbimycin A-pretreated cells, suggesting that the inhibitor acts selectively on tyrosine kinase. Anti-IgM failed to stimulate herbimycin A-pretreated cells to induce increases in inositol phospholipid metabolism or increased [Ca2+]i, whereas aluminum fluoride-induced metabolism was not altered. Moreover, membrane IgM density as revealed by flow cytometry was not changed by herbimycin A. These results indicate that tyrosine kinase(s) participates in the coupling of an Ag receptor cross-linkage to phospholipase C activation through a phosphorylation event in B lymphoma cells.

Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins.

We have recently demonstrated that an IgG3 rheumatoid factor (RF) monoclonal antibody (mAb), clone 6-19, derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice, is able to generate cryoglobulins via a non-immunological IgG3 Fc interaction, and to induce an acute glomerulonephritis associated with cryoglobulinemia. Using this experimental model, we have characterized the glomerular lesions induced by the 6-19 RF monoclonal cryoglobulin, in particular the ultrastructural localization of the cryoglobulin deposits. Although their initial localization was confined to the mesangium, the 6-19 cryoglobulins were progressively accumulated in the subendothelial spaces of glomerular capillary walls, leading to the formation of glomerular lesions resembling the "wire-loop" lesion characteristically described for lupus nephritis. In addition, we have found that identical glomerular "wire-loop" lesions were induced by the 6-19-J558 hybrid antibody, composed of the 6-19 gamma 3 heavy chain and J558 lambda 1 light chain, which loses the RF activity, but retains the cryoglobulin activity. These results strongly suggest that the direct deposition of IgG3 cryoglobulins by itself, without involvement of immune complex formation, results in the generation of the classical "wire-loop" lesion characteristic of lupus nephritis. In addition, we have found that similar "wire-loop" lesions were generated by one anti-DNA mAb derived from (NZB x NZW)F1 hybrid mice, and two of four IgG3 mAb of unknown specificities, derived from MRL/MpJ-lpr/lpr mice. The absence of significant glomerular lesions, in spite of large amounts of cryoglobulins, in mice receiving two IgG3 mAb suggests the importance of physicochemical property of cryoglobulins to provoke glomerular lesions.

IgG3 is the major source of cryoglobulins in mice.

A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3.