A novel, blocking, Fc-silent anti-CD40 monoclonal antibody prolongs nonhuman primate renal allograft survival in the absence of B cell depletion.

CD40-CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti-CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti-CD40 antibody can still promote graft survival. The parent anti-CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC-mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc-silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98-100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well-tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc-silent anti-CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

Smooth muscle cell proliferation but not neointimal formation is dependent on alloantibody in a murine model of intimal hyperplasia.

Transplant coronary artery disease is the pre-eminent cause of late cardiac allograft failure. It is primarily characterized by a concentric intimal hyperplasia, which we designate transplant intimal hyperplasia (TIH). Although the pathogenesis of TIH is predominately immune driven, the specific role of alloantibodies in the disease process remains undefined. In this study we investigated the contribution of alloantibodies to the development of TIH in a murine model. Orthotopic, carotid artery transplantation was performed between B10A(2R) (H-2(h2)) donor mice and B-cell deficient muMT(-/-) knockout or wild-type C57BL/6 (H-2(b)) recipients in the absence of immunosuppression. Grafts were harvested at 35 days and subjected to planimetry and immunohistochemistry. Alloantibodies were detectable in wild-type recipients within 7 days of transplantation and recipients developed marked TIH at 35 days. Allografts harvested from B-cell deficient recipient mice also developed TIH, which was comparable in severity with wild-type recipients. However, whereas allografts from wild-type recipients showed marked intimal smooth muscle cell (SMC) proliferation, the neointima in B-cell deficient recipients lacked SMCs. Post-transplantation administration of anti-donor serum to muMT(-/-) recipients restored neointimal SMC population but did not influence the severity of TIH. Significant neointimal formation occurs in the absence of alloantibodies but lacks a SMC component. Therefore, SMC migration and proliferation is antibody dependent.

Pulmonary inflammation induced by a recombinant Brugia malayi gamma-glutamyl transpeptidase homolog: involvement of humoral autoimmune responses.

BACKGROUND: A major allergen from the lymphatic filarial parasite Brugia malayi implicated in the pathogenesis of tropical pulmonary eosinophilia (TPE) has recently been cloned and identified as the homolog of the membrane-bound mammalian enzyme gamma-glutamyl transpeptidase (gamma-GT). Patients with acute TPE show autoreactive antibodies against endogenous gamma-GT from the pulmonary epithelium. MATERIALS AND METHODS: Recombinant B. malayi gamma-GT, alone or adsorbed to aluminium hydroxide (AL), was used in a BALB/c mouse model to analyze its antigenic/allergenic potential, its potential to induce pulmonary inflammation, and its capacity to induce autoreacting antibodies. RESULTS: Mice immunized with B. malayi gamma-GT showed significant levels of gamma-GT-specific IgG1, IgG2a, IgG3, IgA, IgE antibodies, and mild blood eosinophilia, even in the absence of adjuvant. Intranasal challenge with B. malayi gamma-GT induced peribronchial and perivascular inflammation characterized by a mixed infiltrate of lymphocytes, neutrophils, eosinophils, and macrophages. Both IL-4 and IFN-gamma were detected in the peripheral blood and in the bronchoalveolar lavage fluid of immunized and intranasally challenged mice. Histological analysis of murine lungs using affinity-purified antibodies from mice immunized with the parasite's gamma-GT revealed the presence of autoimmune antibodies against pulmonary epithelium. Western blot analysis identified the 55 kDa heavy chain subunit of the murine gamma-GT as the target of autoreactive/crossreacting antibodies. CONCLUSION: Our data from the in vivo mouse model demonstrate the potent allergenicity/antigenicity of B. malayi gamma-GT, and its capacity to induce pulmonary inflammation upon intranasal challenge. This leads to breakdown of tolerance against endogenous murine gamma-GT. Thus, humoral autoimmunity against the airways epithelium may contribute to the pathogenesis of TPE.

Aromatic quinolinecarboxamides as selective, orally active antibody production inhibitors for prevention of acute xenograft rejection.

The prevention of xenograft rejection is substantially dependent on inhibiting antibodies (Ab) produced by B-cells independently of T-cell signals (TI-1). Due to their ubiquitous biochemical mechanisms of action, the immunosuppressants currently employed not only fail to discriminate between B- and T-cells but also have a narrow therapeutic window and, thus, their prolonged use in complex immunosuppressive regimens is problematic. By capitalizing on the target enzyme-bound (DHODH) structure 1b of one of these compounds, leflunomide, and modulating part of its multiple mechanisms of action to gain selectivity, the quinoline-8-carboxamide 3 was designed as a potentially weak enzyme inhibitor but effective immunosuppressant. Compound 3 fulfilled the mechanistic criteria set and had 10-fold B-cell over T-cell selectivity. Its pyridyl analogue 4 was found to be a highly potent and selective B-cell immunosuppressant with a 75-fold selectivity for B- over T-cells (as judged by the MLR data) and no general cytotoxicity at concentrations up to 160-fold higher than those required to inhibit B-cells. In the mouse, 4 effectively blocked TI-1 Ab production and suppressed Ab-mediated xenograft rejection in a xenotransplantation model under a once-daily dosing regimen, with efficacy down to 0.3 mg/kg/day po. These are the first data demonstrating the feasibility of the development of drugs specific for impeding Ab production.

Allergen-derived long peptide immunotherapy down-regulates specific IgE response and protects from anaphylaxis.

To evaluate a long peptide-based allergy vaccine in a murine model, CBA/J mice were sensitized with low dose alum-adsorbed phospholipase A2 (PLA2), a major bee venom allergen. Presensitized mice were treated by daily i.p. injections of a mixture of three long overlapping peptides (44- to 60-mer) spanning the entire PLA2 molecule (100 microg/peptide) for 6 consecutive days. This therapeutic approach induced a sharp drop in PLA2-specific IgE, an increase in specific IgG2a, and a marked T cell hyporesponsiveness. T cell cytokine secretion was characterized by a shift from a Th2 to a Th1 profile. Prophylactic treatment of naive mice with long peptides prior to sensitization with PLA2 induced a comparable modulation of B and T cell responses. Upon i.p. challenge with native PLA2, presensitized mice treated with the long peptide mixture were fully protected from anaphylaxis. This indicated that allergen-derived long overlapping peptides were safe and able to modulate an established Th2 response or to prevent its development. Furthermore, long peptide-based immunotherapy provided clinical protection against anaphylaxis, thus appearing as a promising approach of the therapy of allergic diseases.

Anti-interleukin 5 but not anti-IgE prevents airway inflammation and airway hyperresponsiveness.

The role of IL-5 and allergen-specific IgE in the development of eosinophilic airway inflammation and airway hyperresponsiveness (AHR) was investigated in a murine model. BALB/c mice were sensitized to ovalbumin (OVA) by intraperitoneal injection on Days 1 and 14, followed by airway challenge with OVA on Days 28 and 29. Anti-IL-5 (TRFK-5) or anti-IgE (antibody 1-5) was administered before each airway challenge. Sensitized and challenged mice developed increased OVA-specific IgE serum levels, Th2 cytokine production by peribronchial lymph node (PBLN) cells, increased numbers of eosinophils (predominantly located in the peribronchial regions of the lungs), and increased airway responsiveness to methacholine (MCh). Anti-IgE treatment significantly decreased serum anti-OVA IgE levels and prevented the development of anaphylaxis but failed to affect T cell function, eosinophil airway infiltration, and AHR in sensitized and challenged mice. In contrast, treatment with anti-IL-5 antibody did not affect B cell (Ig serum levels), T cell (cytokine production), or mast cell function (immediate cutaneous reactivity) but completely inhibited development of eosinophilic lung inflammation and AHR. These data identify IL-5-mediated eosinophilia as a major target for development of AHR in this model, with little effect resulting from neutralization of IgE.

Suppression of immediate and late responses to antigen by a non-anaphylactogenic anti-IgE antibody in a murine model of asthma.

Eosinophils are recruited to the airways during allergic reactions, but animal models have shown that their mere presence is not sufficient for the development of bronchopulmonary hyperreactivity. Other factors, such as immunoglobulin (Ig)E, seem to be required. Using mice selected for the production of large amounts of IgE, the effects of antibody neutralization of IgE on antigen-induced lung recruitment of eosinophils and induction of bronchopulmonary hyperreactivity and of other indicators of inflammation were studied. A monoclonal non-anaphylactogenic rat anti-mouse IgE (mAb1-5), given within 24 h of the challenge with antigen, reduced tissue eosinophilia, the recruitment of IgE-bearing cells identified as basophils, mucous cell metaplasia, anaphylactic bronchoconstriction and bronchopulmonary hyperreactivity. mAb1-5 inhibited interleukin (IL)4 titres in the bronchoalveolar lavage fluid, but not those of I1-5. Inhibition by mAb1-5 may result from competitive displacement of immunoglobulin E from its different receptors, thus preventing cell stimulation. Moreover, the inhibition of the massive recruitment of immunoglobulin E-bearing basophils into the lungs within hours after challenge and of interleukin4 production by mAb1-5 may be important factors leading to the reduction of pulmonary eosinophilia and bronchopulmonary hyperreactivity. Thus, immunoglobulin (Ig)E and allergic IgE-bearing cells seem to play an essential role in the initial development of the late allergic airway responses.

Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro.

The recombinant form of the extracellular domain of the IL-4 receptor (sIL-4R) is a potential candidate to neutralize IL-4; however, murine sIL-4R displayed both antagonistic and agonistic activity in vivo. Here we show that human recombinant sIL-4R induced the formation of complexed IL-4 in supernatants of activated T cells in a dose-dependent manner as measured by newly developed enzyme-linked immunosorbent assays. These IL-4/sIL-4R complexes liberated free IL-4 even after prolonged culturing. In contrast, in the absence of exogenously added sIL-4R, free IL-4 was rapidly consumed or proteolytically degraded in cultures of activated T cells. Thus, no IL-4 bioactivity could be determined in supernatants of T cells activated in the presence of IL-4 for 6 days. In contrast, the same cultures carried out in the presence of sIL-4R showed marked IL-4 bioactivity. While low concentrations of sIL-4R enhanced IL-4-driven inhibiton of IFN-gamma production by activated T cells, higher concentrations neutralized IL-4. Together, human sIL-4R, besides its activity as an antagonist to IL-4, also possesses protective and agonistic functions for IL-4, which may be relevant for clinical studies aiming to neutralize IL-4 in vivo.

Detection of and discrimination between total and free human interleukin-4 and free soluble interleukin-4 receptor by ELISA.

Interleukin-4 (IL-4) signaling is initiated by binding of IL-4 to the high-affinity IL-4 receptor alpha-chain and subsequent interaction with the common gamma-chain. Soluble forms of the extracellular domain of the alpha-chain (sIL-4R) were shown to be present in biological fluids and, dependent on the concentration, enhance or inhibit IL-4 activity by forming IL-4/sIL-4R complexes. To discriminate between free and potentially active IL-4 from the inactive and complexed form, we have established a set of new ELISA systems for the measurement of human IL-4 in its distinct forms. To select suitable pairs of anti-IL-4 antibodies, a chequerboard interference analysis with six highly-selective human IL-4 specific monoclonal antibodies was performed. For the determination of total IL-4, a monoclonal capture antibody was used that binds IL-4 outside the binding site of the IL-4R alpha-chain. Another antibody recognizing an epitope of the alpha-chain binding site was chosen for the detection of free IL-4. The binding of this antibody was inhibited in a dose-dependent fashion by recombinant sIL-4R. Assays for both total and free IL-4 exhibited a sensitivity of 8 pg/ml and a dynamic range up to 1000 pg/ml. Human sIL-4R was detected by two monoclonal antibodies directed against different epitopes. This ELISA was inhibited by recombinant IL-4 suggesting the measurement of predominantly free sIL-4R. Complexes between soluble IL-4R and IL-4 were detected by a monoclonal anti-sIL-4R antibody in combination with an anti-IL-4 antibody. When supernatants of activated T cells were analyzed, the majority of the IL-4 was in free form. The amount of complexed IL-4 was low as indicated by the fact that most of total IL-4 could be detected as free IL-4. Although values obtained for complexed IL-4 correlated with the difference between total and free IL-4, precise values could not be determined, presumably due to the dynamic nature of the complex between the two proteins. We suggest that the ability to quantitate total and free IL-4 in combination with sIL-4R may provide a new insight of the role that IL-4 plays in different pathophysiological conditions.

Frequencies of T cells expressing interleukin-4 and interleukin-5 in atopic asthmatic children. Comparison with atopic asthmatic adults.

T-cell-derived cytokines have been implicated in the pathogenesis of asthma and it has been suggested that Th2-type cytokines (interleukin-4 [IL-4], interleukin-5 [IL-5]) are pivotal in the allergic inflammation. However, there are little data on human cytokine production by individual T cells at the protein level, in particular in asthmatic children. In this study we analyzed the cytokine production at the single cell level in peripheral blood from mild atopic asthmatic (AA) children and adults and age-matched atopic nonasthmatic (AN) and nonatopic nonasthmatic (NN) control subjects (n = 9 in each group) using the technique of intracellular cytokine detection by flow cytometry. Comparing asthmatic children with atopic and nonatopic control subjects, an increased percentage of IL-5-producing T cells (AA: median 4.9% [range 1.1 to 8.9%]; AN: 0.3% [0.2 to 0.9%], p = 0.003; NN: 0.4% [0.1 to 3.8%], p = 0.001) was detectable, with a positive correlation to the number of peripheral eosinophils and to bronchial hyperresponsiveness. The frequency of IL-4-producing T cells was increased in both atopic groups compared with nonatopic controls (AA: 1.2% [0.2 to 2.6%], p = 0.011; AN: 0.8% [0.4 to 3.7%], p = 0.007; NN: 0.4% [0.2 to 0.9%]) with a positive correlation to total IgE concentration. In adults there were no differences in IL-5- or IL-4-producing T cells between all three groups. A substantial proportion of T cells coproducing IL-4 and IL-5 was not detectable in children and adults. These findings indicate that in asthmatic children the frequencies of Th2-type-producing T cells are increased and that expression of IL-4 and IL-5 is regulated independently.

Inhibition of human airway sensitization by a novel monoclonal anti-IgE antibody, 17-9.

We investigated the effect of a novel mouse IgG2b nonanaphylactogenic anti-human IgE antibody, 17-9, on allergen and histamine responses in passively sensitized human airways in vitro to determine the specific contribution of IgE to the sensitization process. Bronchial rings were sensitized with serum containing high levels of allergen-specific IgE (Dermatophagoides farinae), or with a hapten-specific chimeric humanized IgE (JW8). There was a concentration-dependent contraction of serum-sensitized bronchial rings to D. farinae (517 +/- 188 mg tension at 10 U/ml, n = 8) that was not observed in nonsensitized controls. This response was practically abolished when tissues were sensitized in the presence of 100 microg/ml anti-IgE antibody 17-9 (54 +/- 20 mg). In tissues sensitized with the anti-NIP IgE, JW8, there was a concentration-dependent contraction to the specific antigen NIP-BSA (560 +/- 154 mg at 0.3 microg/ml, n = 5) that was not observed in nonsensitized control subjects and that was substantially inhibited when 17-9 was present in the sensitization buffer (124 +/- 109 mg). The inhibition with 17-9 was specific, as pretreatment with a non-IgE-specific IgG2b antibody did not affect allergen responses. Potency and maximal contractions to histamine in serum-sensitized tissues were significantly elevated compared with nonsensitized controls; this was not affected by the presence of 17-9 during sensitization (pEC50 = 5.1 +/- 0.2 versus 5.0 +/- 0.3 in tissues sensitized in the absence of 17-9). In tissues sensitized with JW8 there was no significant increase in responsiveness to histamine. We conclude that allergen responses in sensitized human airways are dependent on IgE levels in the sensitizing serum while nonspecific (hyper)responsiveness depends on serum factors other than IgE. Nonanaphylactogenic anti-human IgE antibodies effectively inhibit allergen responses of human airways in vitro but may not affect other factors inducing hyperresponsiveness.

Glucocorticoids inhibit human antigen-specific and enhance total IgE and IgG4 production due to differential effects on T and B cells in vitro.

Although anti-inflammatory properties of glucocorticoids (GC) are well documented, their activity in allergic diseases is still controversial. Recently, it has been reported that GC can increase, both in vivo and in vitro, the polyclonal production of total IgE. In this study we investigated the effects of GC on the antigen (Ag)-specific IgE response in a human in vitro system with peripheral blood mononuclear cells or B cells of bee venom-sensitized individuals that allows the production of bee venom phospholipase A2 (PLA)-specific IgE and IgG4 antibodies (Ab). PLA-specific Ab were induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand (sCD40L) in the presence of interleukin (IL)-4. Indeed, dexamethasone and prednisolone enhanced the formation of total IgE and IgG4 in PBMC, while the production of PLA-specific IgE and IgG4 Ab was selectively inhibited in a dose-dependent manner. The suppressive effect of GC was mediated during Ag-specific stimulation and T cell-B cell interaction. This was due to GC suppressing specific T cell proliferation and cytokine production, whereas neither allergen-specific nor total IgE and IgG4 production by sCD40L/IL-4-stimulated pure B cells was affected. In contrast to GC, cyclosporine A inhibited both total and PLA-specific IgE and IgG4 secretion in peripheral blood mononuclear cells and B cell cultures. Further experiments showed that increase in nonspecific total isotype response resulted from inhibition of IL-4 uptake by cells other than B cells and sufficient availability of IL-4 to B cells for isotype switch and synthesis. Furthermore, demonstration of opposite regulatory effects of GC on specific and total isotype formation in vitro, including the inhibition of allergy-relevant Ag-specific IgE response, may contribute to a better understanding of apparently controversial observations, and explain why most allergic patients benefit from GC therapy.

Opposite role for interleukin-4 and interferon-gamma on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells.

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4+ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA+ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30+ cells were only found in CD4+ and CD8+ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-gamma. By contrast, LAG-3 expression was strongly up-regulated in CD4+ and CD8+ T cells from cultures primed with IL-12, which developed into high numbers of IFN-gamma producers. The addition of a neutralizing anti-IFN-gamma antibody to IL-12-primed CD4+ T cell cultures virtually abolished the development of LAG-3-expressing CD4+ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-gamma during the lineage commitment of human naive T cells.

Demonstration of the therapeutic potential of non-anaphylactogenic anti-IgE antibodies in murine models of skin reaction, lung function and inflammation.

BACKGROUND: Allergies and allergic asthma are believed to be mediated by allergen-specific IgE antibodies. We have investigated the therapeutic potential of inhibiting endogenous IgE by a non-anaphylactogenic anti-mouse IgE antibody 1-5 with respect to its effects on antigen-induced skin reaction, lung function changes and lung inflammation in mice. METHODS: Mice were immunized with benzylpenicillinoyl-KLH or ovalbumin, and antigen-mediated skin reaction, bronchoconstriction, bronchopulmonary hyperresponsiveness (BHR) and lung eosinophilic inflammation determined in anti-IgE 1-5-treated versus untreated animals. RESULTS: Application of anti-IgE 1-5 inhibited (by 90%) the serum IgE and, 3-4 days after onset of treatment, blocked the antigen-induced skin reaction. Furthermore, the antibody also inhibited (by 90%) the antigen-induced infiltration of eosinophils into the lung. This latter effect seems to be mediated by blocking the IgE-CD23 interaction and indicates that lung eosinophilic inflammation also depends on IgE. Moreover, when applied to rats passively sensitized with mouse IgE, antibody 1-5 inhibited the antigen-induced bronchoconstriction. A similar effect could be seen in actively immunized mice, where antibody 1-5 was able to inhibit (by 70%) the ovalbumin-induced bronchoconstriction as well as BHR. CONCLUSIONS: In summary, non-anaphylactogenic anti-IgE antibodies can markedly inhibit IgE levels and IgE-mediated allergic reactions. Since bronchoconstriction, BHR and lung eosinophilic inflammation can be suppressed, such antibodies may be attractive principles for the treatment of allergic asthma.

Induction and differential regulation of bee venom phospholipase A2-specific human IgE and IgG4 antibodies in vitro requires allergen-specific and nonspecific activation of T and B cells.

Investigations on the mechanisms of IgE regulation in vitro have been conducted thus far in systems that allow the synthesis of total rather than specific IgE. To study the regulatory prerequisites of antigen-specific IgE antibody production, we have established a culture system that allows the generation of bee venom phospholipase A2-specific IgE and IgG4 antibodies. Allergen-specific IgE was induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand in the presence of IL-4. Additional stimulation of T cells through the CD2 activation pathway by two different anti-CD2 monoclonal antibodies enhanced both the allergen-specific and the total IgE and IgG4 responses. An optimal amount of allergen (0.1 ng/ml) resulted in the induction of both allergen-specific IgE and IgG4 antibodies. Higher antigen doses reduced allergen-specific antibodies and enhanced total isotype production. This differential regulation of allergen-specific and total isotypes reflects different allergen dose-dependent mechanisms in specific and polyclonal activation of T and B cells. Although both isotypes require IL-4 for initial induction, opposite regulatory effects by T cells were observed for IgE and IgG4 antibody expression. In peripheral blood mononuclear cell cultures stimulated with soluble CD40 ligand, IL-4, and phospholipase A2, stimulation of T cells with higher amounts of anti-CD2 enhanced IgG4 in parallel to increased IL-2 and interferon-gamma secretion but inhibited IgE synthesis. These results provide evidence for differential regulation of allergen-specific and total IgE and IgG4 by antigen concentration and demonstrate the pivotal role of T cells controlling the synthesis of the IgE and IgG4 antibody isotypes.

The effect of intravenous administration of a chimeric anti-IgE antibody on serum IgE levels in atopic subjects: efficacy, safety, and pharmacokinetics.

CGP 51901 is a non-anaphylactogenic mouse/human chimeric anti-human IgE antibody that binds to free IgE and surface IgE of IgE-expressing B cells but not to IgE bound to high affinity IgE receptors (Fc epsilonR1) on mast cells and basophils or low affinity IgE receptors (Fc epsilonR2) on other cells. A phase 1 double-blind, placebo-controlled, single dose study with doses of 3, 10, 30, and 100 mg of CGP 51901 was conducted in 33 pollen-sensitive subjects who had raised levels of serum IgE and received either intravenous CGP 51901 or placebo. The administration of CGP 51901 was well tolerated and resulted in a decrease of serum free IgE levels in a dose-dependent manner, with suppression after 100 mg of CGP 51901 reaching > 96%. Time of recovery to 50% of baseline IgE correlated with the dose of administered antibody and ranged from a mean of 1.3 d for the 3 mg to 39 d for the 100 mg dose. Total IgE, comprised of free and complexed IgE, increased as stored and newly synthesized IgE bound to CGP 51901. Complexed IgE was eliminated at a rate comparable with the terminal half-life of free CGP 51901 (11-13 d at all doses). Only one subject showed a weak antibody response against CGP 51901. We conclude that the use of anti-human IgE antibody is safe and effective in reducing serum IgE levels in atopic individuals and provides a potential therapeutic approach to the treatment of atopic diseases.

Role of IL-13 in CD4 T cell-dependent IgE production in atopy.

IgE isotype switching of human B cells requires physical interaction of T and B cells via surface molecules, and either IL-4 or IL-13 secreted by T cells. In this study we analyzed the role of IL-4 versus IL-13 in IgE production in atopy. We found that peripheral blood mononuclear cells (PBMC) from atopic individuals but not from nonatopic subjects secreted IgE without addition of IL-4 or IL-13, if T and B cells were simultaneously activated by anti-CD3 mAb and soluble CD40L, respectively. IgE production by atopic PBMC was dependent on endogenously secreted IL-4 and IL-13, since it could be blocked by a combination of anti-IL-4 plus anti-IL-13 antibodies. No differences in the B cell compartment of nonatopics and atopics were detectable, since PBMC from both donor populations secreted comparable amounts of IgE, if only the B cells were activated by soluble CD40L plus either exogenous IL-4 or IL-13. Further phenotypic analysis of T cells from atopics revealed that activated CD4+45RO- secreted IL-4 but no IL-13, whereas CD4+45RO+ memory T cells secreted low amounts of IL-4, but large amounts of IL-13. Accordingly, prolonged activation of native CD4+45RO- T cells in vitro induced expression of CD45RO, and strongly favored secretion of IL-13 rather than IL-4. Addition of exogenous IL-4 during activation further increased both IL-4 and IL-13 production to a similar degree. However, the potential of CD4 T cells from atopics to deliver contact-dependent activation signals to B cells and to induce IgE production (in the absence of soluble CD40L) increased with prolonged activation, and coincided with IL-13 rather than IL-4 production. Under similar conditions, CD8 effector cells secreted IL-13 but no IL-4, did not express CD40L, and could not help Ig(E) production by B cells. These results suggest that, in atopy, persistently stimulated CD4+45RO+memory/effector T cells provide contact-dependent activation signals to B cells, and that these cells may induce IgE switching largely via secretion of IL-13.

A subset of CD8+ T cells from allergic patients produce IL-4 and stimulate IgE production in vitro.

BACKGROUND AND OBJECTIVE: A subset of IL-4 producing CD8+ T cells was recently identified in HIV patients. Based on these findings we examined whether IL-4 producing CD8+ T cells would also be present in allergic patients and what would be the functional relevance of this T-cell population. METHODS: We investigated the role of CD8+ T cells in IgE production of allergic diseases by analysing the cytokine profile of individual CD4+ and CD8+ T cells. RESULTS: In allergic patients about twice as many CD4+ T cells and six times as many CD8+ T cells produced IL-4 as in non-allergic controls. In contrast the frequency of IFNgamma+ T-cell subsets did not significantly differ between the allergic and non-allergic individuals. The frequency of IL4+ CD8+ T cells correlated with the level of serum IgE. Coculture experiments with T cells or purified CD8+ T cells together with autologous B cells indicated that CD8+ T cells enhanced IgE in vitro, but not IgM production, even when they were physically separated from B cells. This effect could be partially blocked by addition of an IL-4 binding protein, a soluble IL-4 receptor indicating that IL-4 is involved in CD8+ T-cell mediated IgE production. CONCLUSIONS: These data indicate a positive role of IL-4 secreting CD8+ T cells in IgE regulation in allergic patients.

Therapeutic potential of anti-IgE antibodies.

Anti-IgE antibodies directed against the Fc epsilon RI-binding region on IgE inhibit binding of IgE to IgE receptors without inducing mediator release from IgE sensitized cells. In mice these antibodies selectively reduce serum IgE, inhibit antigen induced skin reactions, cytokine production by lung Th2 cells, and pulmonary eosinophil infiltration. Clinical trials in humans reveal that such antibodies are well tolerated and reduce rhinitis symptoms and early and late phase bronchoconstriction responses. Thus interruption of the allergic cascade at the IgE antibody level with non-anaphylactogenic anti-IgE antibodies is effective and represents an attractive intervention for the treatment of allergic diseases.