Jan Ceuppens

Anti-tumor necrosis factor treatment restores the gut barrier in Crohn’s disease

OBJECTIVES:A primary defect of the tight junctions and, hence, increased intestinal epithelial permeability has been proposed as a basic pathogenic event in Crohns disease. Challenge of the mucosal immune system by the commensal gut flora would then result in chronic inflammation. Alternatively, increased permeability could be the result of inflammation. Our aim was to study intestinal permeability in refractory Crohns disease before and after treatment with monoclonal chimeric antibodies directed against tumor necrosis factor (TNF) to investigate whether the abnormal permeability persists after control of inflammation.METHODS:Twenty-three patients with active Crohns disease were evaluated before and 4 wk after a single infusion of 5 mg/kg infliximab. Intestinal permeability was studied by measurement of urinary excretion of 51Cr-EDTA after oral intake.RESULTS:The increased permeation of 51Cr-EDTA through the small intestine (1.63% interquartile range [IQR] 1.06–2.07) and the overall permeation (3.27% IQR 2.40–4.38) before therapy decreased significantly after infliximab infusion to values (1.04% IQR 0.74–1.54 and 2.42% IQR 2.03–2.80, respectively) in the range of those found in normal volunteers (1.12% IQR 0.85–1.58 and 2.28% IQR 1.88–2.86, respectively).CONCLUSION:Inhibiting the proinflammatory cytokine tumor necrosis factor dramatically reduces gut inflammation and largely restores the gut barrier in Crohns disease. Our data confirm the central role of TNF in gut barrier modulation in inflammatory conditions in vivo.

Original article: Sinonasal pathology in nonallergic asthma and COPD: ‘united airway disease’ beyond the scope of allergy

Background:u2002 In contrast to the epidemiological and clinical association between allergic rhinitis and asthma, upper airway inflammation is less characterized in patients with nonatopic asthma and virtually unexplored in chronic obstructive pulmonary disease (COPD). Here, sinonasal pathology is studied in patients with allergic asthma, nonallergic asthma and COPD.

Subset markers of CD8(+) cells and their relation to enhanced cytotoxic T-cell activity during human immunodeficiency virus infection

Using fresh whole blood or isolated lymphocytes, the activity ofin vivo generated cytotoxic T-lymphocytes (CTL) was measured as the OKT3-specific lysis of HL-60 targets, in a cross-sectional study of 53 HIV(+) patients. CTL activity in the entire HIV(+) group was two to three times higher than in HIV(−) controls, with WHO stage 3 (=pre-AIDS) patients showing the highest cytolytic function. The whole-blood CTL assay was validated and its practical and theoretical advantages are discussed. Within the CD8(+) cells, the number and proportion of the CD45RO(+) “memory” subset were significantly increased in HIV(+) subjects. The HLA-DR(+) subset rose most spectacularly in the asymptomatic stage of the infection, while the CD38(+) subset was the only one still significantly rising between the pre-AIDS and the AIDS stage. CTL activity was most closely correlated with T8 cells expressing the CD38 marker. In the context of CTL, CD38 thus seems to reflect activation rather than immaturity. Lymphocytes from HIV(+) subjects with a high OKT3-specific lytic capacity also destroyed normal lymphoblasts to a significant extent, pointing to their possible involvement in an autodestructive process. Our data thus suggest the importance of T8 cytolytic function and/or T8 subtyping in the immunopathogenesis and the prognosis of HIV infection.

Type III IFN‐λ mRNA expression in sputum of adult and school‐aged asthmatics

Background The increased susceptibility of asthmatics to rhinovirus infection has recently been related to deficient IFN‐λ1 (IL‐29) and IFN‐λ2/3 (IL‐28) production by bronchial epithelial cells and macrophages.

Caspase Activation and Apoptosis Induction by Adalimumab: Demonstration In Vitro and In Vivo in a Chimeric Mouse Model

Background: Adalimumab is a fully human monoclonal antibody to tumor necrosis factor (TNF), which was recently introduced as a therapy for Crohns disease and rheumatoid arthritis. Besides neutralization, induction of apoptosis of monocytes/macrophages and T cells is thought to be an important mechanism of action of the anti‐tumor necrosis factor monoclonal antibody infliximab, at least in Crohns disease therapy. Aim: To study caspase activation and the induction of apoptosis by adalimumab and the effect of a caspase inhibitor in vivo. Methods: For in vitro studies, THP‐1 cells (human monocytic cell line) were incubated with adalimumab, infliximab, or human immunoglobulin G, and annexin V + propidium iodide, Apo2.7, and 7‐amino actinomycin‐D were used to study apoptosis on the cell membrane, mitochodrial, and DNA level, respectively. Active caspase‐3 was detected by intracellular staining. For in vivo studies, a chimeric human‐mouse model was used, in which THP‐1 cells were injected intraperitoneally in SCID‐Beige mice followed by treatment with adalimumab, infliximab, or human immunoglobulin G. Effects of a pan‐caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyketone on apoptosis induction were evaluated. Results: In vitro analysis revealed that apoptosis could be induced in THP‐1 cells by both adalimumab and infliximab. Activation of caspase‐3 after incubation with adalimumab was demonstrated by intracellular staining. In addition, in the chimeric mouse model, a higher percentage of residual THP‐1 cells were apoptotic, and lower cell numbers were recovered in the adalimumab‐ or infliximab‐treated mouse. Apoptosis induction by adalimumab could be abrogated through in vivo pretreatment of mice with the pan‐caspase inhibitor. Conclusions: Adalimumab, besides neutralizing tumor necrosis factor, also induces apoptosis of transmembrane tumor necrosis factor‐positive THP‐1 cells by activating intracellular caspases. This activity is likely to be important for the clinical effect of this biodrug.

Essential Role for CD40 Ligand Interactions in T Lymphocyte-Mediated Modulation of the Murine Immune Response to Pneumococcal Capsular Polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4+ T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4+ T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4+ T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4+ T lymphocytes. CD8+ T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8+ T lymphocytes on the anti-caps-PS immune response. CD4+ T lymphocyte-depleted murine spleen cells, leaving a B and CD8+ T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.

T lymphocyte dependence of the antibody response to ‘T lymphocyte independent type 2’ antigens

With regard to their capacity for antibody induction, antigens can be classified as either T lymphocyte dependent (TD) or as T lymphocyte independent (TI).1 In the immune response to TD antigens, T lymphocyte help to B lymphocytes is essential for the regulation of B lymphocyte proliferation, production of immunoglobulins, immunoglobulin class switching, rescue of B lymphocytes from apoptotic death, germinal centre formation, and generation of B lymphocyte memory.2 In contrast to TD antigens, TI antigens induce antibody production without the help of T lymphocytes. TI antigens can be further divided into TI type 1 antigens (TI-1), which are polyclonal B lymphocyte activators (e.g. lipopolysaccharides), and TI type 2 (TI-2) antigens. TI-2 antigens are able to directly stimulate B lymphocytes. However, the antibody response to TI-2 antigens is somehow influenced by T lymphocytes.3 TI-2 antigens do not induce immunological memory and antibodies to TI-2 antigens in humans only develop after the age of 2 years.4,5 n nGenerally, TI-2 antigens are antigens that consist of repetitive biochemical structures such as polymeric protein antigens, trinitrophenyl-ficoll (TNP-ficoll), and dinitrophenyl-ficoll (DNP-ficoll). A clinically important group among the TI-2 antigens are the bacterial capsular polysaccharides.6 Capsular polysaccharides of Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis are responsible for the bacterial virulence and antibodies to capsular polysaccharides provide protection against invasive infections with these bacteria.7 The delay in antibody formation to encapsulated bacteria renders infants and young children highly susceptible to infections with encapsulated bacteria, especially from the ages of 4 to 6 months on, when the placentally derived maternal IgG is metabolized.5 Therefore, children younger than 2 years of age are more at risk for invasive infections caused by encapsulated micro-organisms.8 Children with a persisting defect in the production of antibodies specific for pneumococcal capsular antigens after this age have the so-called ‘specific antibody deficiency with normal immunoglobulins’ (SADNI). They suffer from recurrent pneumococcal infections, although their immunoglobulin and immunoglobulin subclass levels and responses to protein antigens are normal.9–12 It is estimated that 5–10% of the children referred for evaluation of recurrent infections have SADNI and it is therefore highly important to understand the immunological background of the antibody formation against TI-2 antigens.13 In this review we will summarize the current understanding of how T lymphocytes modulate the antibody response against TI-2 antigens.

Persistent IL-10 production is required for glioma growth suppressive activity by Th1-directed effector cells after stimulation with tumor lysate-loaded dendritic cells

Injection of dendritic cells (DC) pulsed with tumor antigens is a novel treatment strategy against malignancies, and aims to elicit anti-tumoral cell-mediated immune responses. We studied the in vitro proliferative responses and cytokine production in T cell cultures after 2 stimulations with autologous DC loaded with tumor lysates derived from glioblastoma multiforme (GBM) cells in the presence of recombinant interleukin (rIL)−6/rIL-12 in the first, and rIL-2/rIL-7 in the second stimulation. After the second stimulation, T cells were co-cultured with glioblastoma (GBM) cells and tumor growth suppression by T cells was assessed using a MTT assay. Although loaded DC induced a significant shift towards T helper cell type 1 (Th1) cytokine production as compared to unloaded DC, persistent interleukin (IL)-10 production by T cells both at the end of 2 stimulations with loaded DC and during the effector phase was also required for their tumor suppressive activity. A stronger glioma growth suppressive activity by T cells stimulated with tumor lysate-loaded DC than by control T cells, cultured with unloaded DC, was seen only if the relative IL-10 production after two stimulations with loaded DC was at least 40% of the IL-10 production after two stimulations with unloaded DC. If less than 40% IL-10 was produced in the experimental condition compared to the control condition, T cells also lost their tumor growth suppressive activity. Addition of rIL-10 during stimulation increased the suppressive activity on tumor cell viability and interferon (IFN)-γ production by T cells that showed Th1 response upon stimulation with loaded DC. The data point towards the production of both IFN-γ and IL-10 by responding effector T cells, and towards an immune modulatory rather than immune suppressive role of IL-10 to generate anti-tumoral effector T cells against GBM.

The human antibody response to pneumococcal capsular polysaccharides is dependent on the CD40-CD40 ligand interaction

Protection against infections with Streptococcus pneumoniae is mediated by antibodies against the capsular polysaccharides (caps‐PS). Here we show that in in vitro experiments CD4+ Tu2004lymphocytes stimulate and CD8+ Tu2004lymphocytes inhibit the human anti‐caps‐PS antibody response. Using antagonistic anti‐CD40 and antagonistic anti‐CD40u2004ligand (CD40L) monoclonal antibodies, we showed that the CD4+ Tu2004lymphocyte‐mediated stimulation is dependent on the CD40‐CD40L interaction. The role of CD40L was further illustrated by the observation that CD4+ Tu2004lymphocytes obtained from a patient with hyper‐IgM syndrome were unable to enhance the immune response to caps‐PS. Furthermore, CD4+ Tu2004lymphocytes from cord blood, which did not express CD40L in response to stimulation with caps‐PS, failed to stimulate the antibody response of adult Bu2004lymphocytes to caps‐PS. These in vitro findings were confirmed by in vivo experiments in which SCID/SCID mice were reconstituted with human mononuclear cells. Furthermore, we showed that caps‐PS induce production of IL‐4, IL‐6, IL‐10, and IFN‐γ, and that thisenhanced production was inhibited by blocking the CD40‐CD40L interaction. This is the first demonstration that the human immune response to caps‐PS, which is markedly regulated by Tu2004lymphocytes, is dependent on the CD40‐CD40L interaction.

CD4+ T Lymphocytes Expressing CD40 Ligand Help the IgM Antibody Response to Soluble Pneumococcal Polysaccharides via an Intermediate Cell Type

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.