CD40 and IgE: synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells.

A novel pathway of IgE-B cell differentiation has been identified. Engagement of the B cell antigen CD40 by F(ab')2 fragments of monoclonal antibody (mAb) 626.1 in the presence of recombinant interleukin 4 (rIL-4) induced intense IgE synthesis, but modest IgG synthesis, by highly purified human B cells. Surface IgE- B cells isolated by cell sorting were induced to produce IgE by mAb 626.1 and IL-4. Thus, IgE synthesis is unlikely to result from expansion of a B cell population precommitted to IgE in vivo. A neutralizing anti-IL-6 antibody strongly, but not completely, inhibited the IgE response. This indicates that autocrine production of IL-6 plays an important amplification role in IgE synthesis triggered by anti-CD40 mAb and IL-4. Although the exact role played by CD40 in IgE responses in vivo remains to be established, this T cell-independent system represents a useful model to characterize the biochemical and molecular events leading to IgE synthesis in human B cells.

Induction of human IgE synthesis requires interleukin 4 and T/B cell interactions involving the T cell receptor/CD3 complex and MHC class II antigens.

The induction of IgE synthesis by IL-4 requires T cells and monocytes, as well as T cell- and monocyte-derived cytokines. Optimal cytokine combinations, however, fail to induce highly purified B cells to secrete IgE, indicating that additional signals are required. We show herein that the induction of human IgE synthesis by rIL-4 requires cognate interaction between the T cell receptor/CD3 complex on T cells and MHC class II antigens on B cells: mAbs directed against these molecules completely blocked IL-4-dependent IgE induction. mAbs against cell adhesion molecules (CD2, CD4, LFA-1) also inhibited IgE synthesis induced by IL-4, confirming that cell-cell contact is necessary for IgE induction. The requirement for cognate T/B cell interaction was further shown by comparing the IgE-inducing ability of two human IL-4-producing alloreactive T cell clones: F6, which recognizes MHC class II antigens on both B cells and monocytes, and A1, which recognizes an HLA-DP-associated epitope expressed on monocytes, but not on B cells. When incubated with B cells and monocytes from a normal donor bearing the appropriate alloantigen, clone F6, but not clone A1, induced vigorous IgE synthesis, although both clones proliferated and secreted IL-4. Taken together, our results suggest that at least two, possibly synergizing, signals are required for the T cell-dependent induction of IgE synthesis by B cells: one signal is delivered by cognate T/B cell interaction, the other by T cell-derived IL-4.

Molecular analysis of the induction of immunoglobulin E synthesis in human B cells by interleukin 4 and engagement of CD40 antigen.

The molecular events leading to immunoglobulin E (IgE) synthesis in human sIgE- B cells stimulated with interleukin 4 (IL-4) and anti-CD40 monoclonal antibody (mAb) 626.1 were analyzed. Anti-CD40 mAb increased the levels of IL-4-induced germline C epsilon transcripts and induced the production of mature C epsilon mRNA. These effects were dependent on the presence of IL-4. Nested primer PCR revealed deletional switch recombination occurring only in B cell stimulated with both IL-4 and anti-CD40 mAb. DNA sequence analysis of switch fragments showed direct S mu/S epsilon joining, without the deletions or duplications within S mu often found in B cells stimulated with IL-4 and Epstein-Barr virus. Analysis of the switch junction map sites showed "hot spots" for recombination within S mu, but not within S epsilon. These findings indicate that IL-4 provides a signal to B cells to induce germline C epsilon transcription and concurrent CD40 engagement induces S mu/S epsilon deletional switch recombination, production of mature C epsilon mRNA, and IgE synthesis.

Human recombinant interleukin 4 induces Fc epsilon R2/CD23 on normal human monocytes.

rIL-4 (B cell stimulatory factor 1) induces the expression of Fc epsilon R2/CD23 on normal human monocytes (Mo). Fc epsilon R2/CD23 induction was detectable both by flow cytometry using anti-CD23 mAbs as well as soluble IgE, and by the immunoprecipitation with CD23-specific mAb or IgE of a 45-kD band from 125I-lactoperoxidase-labeled Mo. Fc epsilon R2/CD23 was fully expressed after a 24-h incubation with rIL-4, and was still detectable after 72 h from the addition of IL-4. This effect was specific, because none of the other rILs tested (IL-1, IL-2, IL-3, IL-5, B cell stimulatory factor 2, granulocyte-macrophage colony stimulating factor, and IFN-gamma) could induce FC epsilon R2/CD23, either alone or in various combinations. No synergism was observed between IL-4 and other ILs. IFN-gamma was not able to inhibit the IL-4-induced expression of Fc epsilon R2/CD23 on Mo, neither when added to the culture together with IL-4, nor when added 36 h earlier.

C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Regulation of immunoglobulin (Ig)E synthesis in the hyper-IgE syndrome.

The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis, and recurrent infections. The mechanisms responsible for hyperproduction of IgE in HIE patients are presently unknown. We investigated whether spontaneous in vitro IgE synthesis by PBMC from seven HIE patients was sensitive to signals (cell adhesion, T/B cell cognate interaction and lymphokines: IL-4, IL-6, and IFN-gamma) known to regulate IgE induction in normals. Our results show that, unlike IL-4 dependent IgE synthesis induced in normals, spontaneous IgE production by PBMC from HIE patients was not blocked by monoclonal antibodies to CD2, CD4, CD3, and MHC class II antigens. Furthermore, antibodies to IL-4 and IL-6 did not significantly suppress IgE production. IFN-gamma had no significant effects on spontaneous in vitro IgE synthesis. To test whether an imbalance in lymphokine production might underlie hyperproduction of IgE in HIE patients, mitogen-induced secretion of IL-4 and IFN-gamma by PBMC was assessed. No significant difference was detected between HIE patients and normal controls. Thus, ongoing IgE synthesis in the HIE syndrome is largely independent of cell-cell interactions and endogenous lymphokines, and is due to a terminally differentiated B cell population, no longer sensitive to regulatory signals.

Regulation of isotype switching.

In the past year, our understanding of the mechanisms that regulate isotype switching has significantly progressed. Activation of germ line transcription by isotype-specific cytokines is emerging as a crucial mechanism for increasing the accessibility of a particular switch region, and targeting switch recombination.

Cytolytic T lymphocytes with natural killer activity in thyroid infiltrate of patients with Hashimoto's thyroiditis: analysis at clonal level.

T Lymphocytes from thyroid infiltrates and peripheral blood (PB) of 3 patients with Hashimoto's thyroiditis (HT) were cloned using a microculture system previously shown to allow the clonal expansion of virtually all PB T lymphocytes from normal individuals. The phenotypic and functional features of a total number of 153 clones from thyroid infiltrates and 206 clones from PB were examined and compared with those of 272 clones derived from normal PB and spleens. The majority of clones derived from thyroid infiltrates of patients with HT had the cytotoxic/suppressor (T8+) phenotype, whereas the majority of clones from PB expressed the helper/inducer (T4+) phenotype. In addition, a consistent proportion (25%) of clones derived from PB of one patient had a phenotype (T3+T4-T8-) that was only occasionally found on clones obtained from PB or spleens of normal subjects. Most clones derived from both PB and thyroid infiltrates of the patients with HT had cytolytic activity, assessed by a lectin-dependent cytolytic assay against the murine P815 tumor cell line. The high frequency of cytotoxic T cells in thyroid infiltrates was related to the increased proportion of T8+ cells, whereas enhanced percentages of cytotoxic cell precursors with T4+ and T3+T4-T8- phenotypes primarily accounted for the high frequency of cytolytic T cells in the PB of the same patients. Many cytolytic T cell clones derived from thyroid infiltrates also had natural killer activity against human K562 and MOLT-4 target cells. These data provide the first functional analysis of T lymphocytes infiltrating the thyroid gland in patients with HT and suggest that the high proportions of cytolytic T cell precursors found in both thyroid infiltrates and PB of these patients may be of importance in determining the tissue damage in thyroid autoimmune disease.

CD14: a bridge between innate immunity and adaptive IgE responses.

Total IgE levels are known to be under genetic control. Linkage studies have indicated that one or more loci on chromosome 5q may control total IgE, as well as asthma and bronchial hyperresponsiveness to non-specific stimuli. Our group has undertaken a systematic analysis of chromosome 5q, and has recently characterized five single nucleotide polymorphisms at position -1619, -1359, -1145, -809, and -159 in the promoter of the gene encoding CD14, the myeloid pattern recognition receptor that is critical for efficient innate immune responses to lipopolysaccharide and bacterial ligands. Individuals homozygous for the three major CD14 haplotypes found in the Children Respiratory Study population (n = 390) were analyzed for serum levels of total IgE and soluble CD14. A strong inverse correlation was found between these two parameters, i.e. carriers of the -1359T/-1145A/-159C haplotype had the highest levels of IgE, and the lowest levels of sCD14. Conversely, carriers of the -1359G/-1145G/-159T haplotype had the highest levels of sCD14 and the lowest IgE values. Our results suggest that genetic variation in CD14, a key gene of innate immunity, may modulate the effects that exposure to bacterial ligands has on the development of Th2 responses.

Role of protein tyrosine kinases and phosphatases in isotype switching: crosslinking CD45 to CD40 inhibits IgE isotype switching in human B cells.

Protein tyrosine kinases and protein tyrosine phosphatases play an important role in the transduction of signals via antigen receptors in T and B cells, and in CD40-dependent B-cell activation. To examine the role of tyrosine kinases and phosphatases in B-cell isotype switching, we examined the effects of the engagement of the transmembrane phosphatase CD45 on the synthesis of IgE induced by IL-4 and anti-CD40 monoclonal antibody (mAb). Crosslinking CD45 to CD40 using biotinylated mAbs and avidin strongly inhibited CD40-mediated IgE synthesis in IL-4-treated human B cells. CD40/CD45 crosslinking did not affect epsilon germline transcription in B cells stimulated with IL-4, but strongly inhibited induction of S mu/S epsilon switch recombination as detected by a nested primer polymerase chain reaction assay. The B-cell src-type tyrosine kinase lyn, which is activated following CD40 engagement, is a potential target for the effects of CD45 observed in our experiments, because CD45/CD40 crosslinking resulted in the inhibition of CD40-mediated lyn phosphorylation and activation. These results suggest an important role for protein tyrosine kinases and phosphatases in CD40-mediated induction of isotype switching to IgE.

Regulation of human IgE synthesis.

Allergic diseases result from the interaction with IgE bound to cell surface receptors. Therefore, rational therapeutic approaches to allergic diseases would be aimed at decreasing IgE and/or at blocking the binding of IgE to effector cells such as mast cells and monocytes. Our investigation of the mechanism of IgE synthesis in man shows that IgE synthesis by peripheral blood mononuclear cells (PBMC) absolutely requires the presence of IL-4 and requires endogenous IL-6, because antibody to IL-6 inhibits IgE production completely. IgE synthesis requires T/B cell contact and involves interactions between B cell surface MHC Class II molecules and T cell surface receptors, as antibodies to both of these cell surface molecules inhibit IgE synthesis. Furthermore, alloreactive T cell clones which are unable to engage the B cell MHC Class II molecules fail to induce IgE synthesis in spite of their ability to secrete IL-4. Studies on the immunoglobulin sites that are involved in IgE binding to high affinity receptors on mast cells and basophils have used recombinant fragments of IgE to block mast cell binding. These studies suggest that a stretch of 76 amino acids which straddles the C epsilon 2 and C epsilon 3 domains is essential for this binding. Parallel studies on IgE binding to low affinity receptors on monocytes and B cells suggest that sequences within C epsilon 3 are involved in this binding. Peptides or analogues that inhibit IgE binding to its cellular receptors may be useful in the treatment of allergic diseases.

Role of CD4 and CCR5 levels in the susceptibility of primary macrophages to infection by CCR5-dependent HIV type 1 isolates.

Macrophages are a preferred target for sexually transmitted human immunodeficiency virus type 1 (HIV-1) isolates that use CCR5 as a coreceptor in combination with CD4. To assess whether the susceptibility of MDMs to infection by an R5 isolate was influenced by CD4 and/or CCR5 expression, levels of membrane CD4 or CCR5 transcripts at the time of infection and ID50 values 15 days postinfection were measured in cultures of primary macrophages infected with HIV-1(10005). To analyze the data, subjects were divided so as to maximize differences in the levels of CD4 or CCR5 expression between groups. Indeed, the difference in CD4 expression between the CD4high (MFI, 16.7 +/- 2.2) and CD4low (MFI, 6.7 +/- 0.7) groups attained high significance (p < 0.005). Of note, susceptibility to infection of MDMs isolated from CD4high donors was strikingly enhanced as compared with CD4low subjects, as shown by a fourfold increase in ID50 titers at day 15 postinfection (p < 0.002). In contrast, no significant difference in ID50 was apparent when the subjects were grouped according to the levels of CCR5 transcripts, even though CCR5 expression in the two groups differed significantly (p = 0.01). These results suggest that, regardless of variations among individuals, the intensity of CD4 expression in macrophages is such that CCR5 levels are above the threshold required for efficient HIV-1 infection. Consistent with this hypothesis, macrophages from three additional donors selected for high CD4 expression and low CCR5 transcripts were found to be highly susceptible to HIV-1 infection.

Immunoglobulin E and its regulators.

Novel paradigms are emerging in the field of IgE regulation. Gene-environment interactions are increasingly considered to be major determinants of the type and amplitude of allergen-induced antibody responses. At the molecular level, the mechanistic connection between transcription and switching has been strengthened by the recent discovery that class switch recombination requires activation-induced deaminase, a novel RNA editing enzyme. These findings will be critical for our understanding of the mechanisms that determine immunoglobulin isotype expression during immune responses.

A, B, O (H) antigenic determinants in superficial transitional cell tumors of bladder.

The initial tumors of 21 patients who earlier had presented with superficial noninvasive transitional cell carcinoma of the bladder and who subsequently underwent cystectomy for invasive disease were examined by the specific red cell adherence test. Eighteen of 21 initial tumors (85.7%) were antigen negative. The time from initial tumor resection ranged from twelve to 168 months (mean thirty-eight months). Loss of blood group antigens indicated enhanced biologic aggressiveness of tumors. However the interval to invasion is sufficiently variable as to preclude the use of this assay for timing of radical cystectomy.

Regulation of IgE synthesis in humans.

At the present time, in no other system are the signals required for isotype switching better understood than in the IgE system. IgE switching is therefore becoming a general model for "directed" isotype switching in mice and humans. The study of IgE regulation has proposed a paradigm of general importance for immunology in the nineties, namely, the requirement for at least two signals in order to trigger the final event, in this case DNA switch recombination to the IgE isotype. The first signal is delivered by a cytokine, IL-4 or IL-13, and is responsible for the choice of the isotype. The second signal is typically delivered upon engagement of CD40 on B cells by the CD40 ligand expressed on T cells, and results in switching and production of IgE. We shall herein discuss the two-signal model for IgE switching in detail, stressing in particular the cross-talk between signals, and the mechanisms responsible for IgE amplification.

[Use of betamethasone in heart surgery]. / Impiego del betametasone in cardiochirurgia.

Clinical experience carried out on 88 high risk patients subjected to open-heart surgery with ECC is reported. The series was subdivided into two groups of patients (A and B) and to these were applied the same surgical and anaesthesiological approaches and the same extracorporeal perfusion technique. In Group B, however, a different method of myocardial protection was employed, in the form of preventive administration of pharmacological doses of betamethasone (3 mg/kg). The results point to a marked reduction in the incidence of postoperative complications in the heart, lungs and bloodstream in Group B compared with Group A.