Rainer Duchmann

Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD)

Hyporesponsiveness to a universe of bacterial and dietary antigens from the gut lumen is a hallmark of the intestinal immune system. Since hyperresponsiveness against these antigens might be associated with inflammation, we studied the immune response to the indigenous intestinal microflora in peripheral blood, inflamed and non‐inflamed human intestine. Lamina propria monocuclear cells (LPMC) isolated from inflamed intestine but not peripheral blood mononuclear cells (PBMC) of IBD patients with active inflammatory disease strongly proliferated after co‐culture with sonicates of bacteria from autologous intestine (BsA), Proliferation was inhibitable by anti‐MHC class II MoAb, suggesting that it was driven by antigen, LPMC from adjacent non‐inflamed intestinal areas of the same IBD patients and PBMC or LPMC isolated from non‐inflamed intestine of controls and patients with IBD in remission, in contrast, did not proliferate, PBMC or LPMC which had been tolerant to bacteria from autologous intestine, however, strongly proliferated after co‐culture with bacterial sonicates from heterologous intestine (BsH). This proliferation was associated with an expansion of CD8+ T cells, increased expression of activation markers on both CD4+ and CD8+ lymphocyte subsets, and production of IL‐12, interferon‐gamma (IFN‐γ), and IL‐10 protein. These results show that tolerance selectively exists to intestinal flora from autologous but not heterologous intestine, and that tolerance is broken in intestinal inflammation. This may be an important mechanism for the perpetuation of chronic IBD.

Induction of cytokine production in naive CD4+ T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells

BACKGROUND & AIMS Murine liver sinusoidal endothelial cells (LSECs) constitutively express accessory molecules and can present antigen to memory Th1 CD4(+) T cells. Using a T-cell receptor transgenic mouse line, we addressed the question whether LSECs can prime naive CD4(+) T cells. METHODS Purified LSECs were investigated for their ability to induce activation and differentiation of naive CD4(+) T cells in comparison with bone marrow-derived antigen-presenting cells and macrovascular endothelial cells. Activation of T cells was determined by cytokine production. LSECs were further studied for expression of interleukin (IL)-12 by reverse-transcription polymerase chain reaction, and the unique phenotype of LSECs was determined by flow cytometry. RESULTS We provide evidence that antigen-presenting LSECs can activate naive CD62Lhigh CD4(+) T cells. Activation of naive CD4(+) T cells by LSECs occurred in the absence of IL-12. In contrast, macrovascular endothelial cells from aorta could not activate naive CD4(+) T cells. The unique functional characteristics of microvascular LSECs together with a unique phenotype (CD4(+), CD11b+, CD11c+, CD80(+), CD86(+)) make these cells different from macrovascular endothelial cells. Furthermore, LSECs did not require in vitro maturation to activate naive CD4(+) T cells. Most importantly, LSECs failed to induce differentiation toward Th1 cells, whereas conventional antigen-presenting cell populations induced a Th1 phenotype in activated CD4(+) T cells. Upon restimulation, CD4(+) T cells, which were primed by antigen-presenting LSECs, expressed interferon gamma, IL-4, and IL-10, which is consistent with a Th0 phenotype. Exogenous cytokines (IL-1beta, IL-12, or IL-18) present during T-cell priming by antigen-presenting LSECs could not induce a Th1 phenotype, but neutralization of endogenously produced IL-4 during T-cell priming led to a reduced expression of IL-4 and IL-10 by CD4(+) T cells upon restimulation. The addition of spleen cells to cocultures of LSECs and naive CD4(+) T cells during T-cell priming led to differentiation of T cells toward a Th1 phenotype. CONCLUSIONS The ability of antigen-presenting LSECs to induce cytokine expression in naive CD4(+) T cells and their failure to induce differentiation toward a Th1 phenotype may contribute to the unique hepatic microenvironment that is known to promote tolerance.

Regulation of endotoxin-induced IL-6 production in liver sinusoidal endothelial cells and Kupffer cells by IL-10

Sinusoidal endothelial cells and Kupffer cells are the first cell populations in the liver that come into contact with gut‐derived endotoxin in portal blood. Although endotoxin concentrations as high as 1 ng/ml are physiologically present in portal blood, no local inflammation is seen. We show that the proinflammatory cytokine IL‐6, which is central to the development of inflammatory reactions in the liver, is produced by sinusoidal endothelial cells and Kupffer cells in response to low concentrations of endotoxin (100 pg/ml to 1 ng/ml). The anti‐inflammatory cytokine IL‐10 down‐regulated endotoxin‐induced IL‐6 release in endothelial and Kupffer cells. Importantly, Kupffer cells secreted IL‐10 after endotoxin stimulation and may therefore participate in the local regulation of inflammation. We have found that IL‐6 secretion in Kupffer cells is tightly regulated by endogenous IL‐10, because increased IL‐6 secretion resulted when neutralizing antibodies to IL‐10 were added to resting and endotoxin‐challenged Kupffer cells. Furthermore, repeated exposure of endothelial cells to endotoxin induced a state of tolerance which resulted in decreased release of IL‐6 in response to a second endotoxin challenge. Our results support the notion that inflammatory reactions in the liver in response to endotoxin are down‐regulated by local release of the anti‐inflammatory cytokine IL‐10 that is produced by Kupffer cells.

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohns disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1).

VCAM‐1 was first identified as an adhesion molecule induced on human endothelial cells (HEC) by inflammatory cytokines such as IL‐1, tumour necrosis factor (TNF), and lipopolysaccharide (LPS). The molecule binds to a variety of leucocytes, including B cells, T cells, basophils, eosinophils and monocytes. Vascular expression of VCAM‐1 has been associated with a number of disease states, including rheumatoid arthritis and vasculitis. The detection of antineutrophil cytoplasmic antibodies (ANCA), especially to proteinase 3 (PR3), has become important in the diagnosis of Wegener’s granulomatosis (WG) and related vasculitides. Recently we were able to demonstrate a direct effect of anti‐PR3 antibodies on neutrophil–endothelial interactions (Blood 1993; 82:1221). Binding of anti‐PR3 antibodies to their antigen translocated into the membrane of HEC leads to an enhanced adhesion of neutrophils via induction of E‐selectin (Clin Exp Immunol 1993; 94:440). The aim of this study was to investigate the effect of anti‐PR3 antibodies on the expression of VCAM‐1. HEC was isolated from umbilical vein and cultured on microtitre plates. After preincubatiion with purified anti‐PR3 antibody, purified control antibodies (SS‐A, SS‐B, RNP) (IgG and F(ab′)2 fragments) or different cytokines (controls), VCAM‐1 was detected on the surface of unfixed HEC by cyto‐ELISA and polymerase chain reaction analysis. Incubation of HEC with anti‐PR3 antibodies led to a marked increase of endothelial VCAM‐1 expression with a peak after 8 h. Incubation with TNF‐α also led to maximal VCAM‐1 expression after 4–6 h (control). Increased adhesion of T lymphocytes to HEC after binding of anti‐PR3 antibodies to their antigen could be confirmed by performing adherence assays. This effect could be inhibited by antibodies to VLA‐4. In conclusion, we have been able to show that cytokine‐like effects of anti‐PR3 antibodies on HEC are not limited to induction of neutrophil adhesion. Anti‐PR3 antibodies may thus contribute to the regulation of T lymphocyte migration from the blood by HEC in ANCA‐related vasculitides.

Bacteria‐Specific T‐Cell Clones are Selective in their Reactivity Towards Different Enterobacteria or H. pylori and Increased in Inflammatory Bowel Disease

In the present study the authors investigated the T‐cell response to different enterobacteria or Helicobacter pylori and tested the hypothesis that the frequency of bacteria‐specific T cells is increased in the intestine of patients with active inflammatory bowel disease (IBD), i.e. Crohn’s disease (CD) and ulcerative colitis (UC). The analysis of a large panel of T‐cell clones (Tc) (n = 888) from peripheral blood, non‐inflamed and inflamed intestine from IBD patients and control individuals shows that both peripheral blood and intestinal T‐cell clones were selectively stimulated by either Salmonella typhimurium Yersinia enterocolitica 03, Escherichia coli or Helcobacter pylori sonicates, that only < 3% of all bacteria‐reactive Tc were crossreactive and that proliferation to bacterial sonicates was inhibited by anti‐MHC class II antibody. In addition, bacteria‐specific Tc from IBD patients were more frequently isolated from inflamed intestine than from peripheral blood (P = 0.0039) or non‐inflamed intestine. These data, from a large number of T‐cell clones, are the first systematic analysis describing the response of individual T cells towards different bacterial species (ssp.). They show that T cells with specificity for distinct antigens or superantigens that are characteristic for a defined bacteria ssp. are present in normal, and increased in inflamed, IBD‐intestine. These bacteria‐specific Tc may play a role in IBD pathogenesis.

HLA-B27-restricted cytotoxic T lymphocyte responses to arthritogenic enterobacteria or self-antigens are dominated by closely related TCRBV gene segments. A study in patients with reactive arthritis.

Identification of the T‐cell receptors (TCR) used by synovial cytotoxic T lymphocytes (CTL) of patients with reactive arthritis (ReA) may be crucial to better understanding the pathogenetic mechanism underlying the HLA‐B27 association ofspondylarthropathies. The authors, therefore, sequenced 25 TCRB chains from HLA‐B27‐restricted CD8+ CTL clones and two clonal lines specific for self‐ or Yersinia enterocolitica antigen isolated from synovial fluids of 3HLA‐B27+ patients with ReA and PBL of one healthy HLA‐B27+ individual. Fourteen non‐HLA‐B27‐restricted CTL served as controls. Both autoreactive and Y. enterocolitica specific HLA‐B27‐restricted CTL used a highlylimited set of VB genes with preferential rearrangement of three closely related VB families (VB 13,14,17), suggesting that these families contain a preferred site for contact with the HLA‐B27 molecule. In addition, the presence of limited TCRBJ usage,limited heterogeneity in CDR3 sequences and dominant clones from individual donors among these CTL indicate that TCRB chain usage is further restricted by a limited set of peptides bound to the HLA‐B27 molecule. Limited TCR usage by SF CTL of ReA patients may lay a basis for therapeutical manipulation of the T‐cell response in the spondylarthropathies.

Pancreatic Panniculitis in an 88-Year-Old Man with Neuroendocrine Carcinoma

Pancreatic panniculitis is a rare complication that occurs in 0.3–3% of patients with pancreatic diseases. Most of the cases reported to date were associated with adenocarcinoma and acute or chronic pancreatitis. We here present an 88-year-old man who was admitted to our institution with a nonfunctional neuroendocrine carcinoma of the pancreas. He subsequently developed pancreatic panniculitis and arthritis. Treatment with octreotide did not have an effect neither on progression of the carcinoma nor on development of new skin lesions. Two months after the diagnosis of pancreatic panniculitis had been made, the patient died from progressing carcinoma. A review of the literature shows that there is no congruent hypothesis for the pathogenesis of pancreatic panniculitis. Vascular damage seems to induce lipolysis by pancreatic enzymes. This eventually leads to fat necrosis. The diversity of disorders that can go along with pancreatic panniculitis suggests an unspecific damage of pancreatic tissue as a first step in the chain of events.

CD4+ and CD8+ clonal T cell expansions indicate a role of antigens inankylosing spondylitis; a study in HLA-B27+ monozygotic twins

Ankylosing spondylitis (AS) is a complex genetic disease in which both MHC and non‐MHC genes determine disease susceptibility. To determine whether the T cell repertoires of individuals with AS show signs of increased stimulation by exogenous antigens, CD4+ and CD8+ T cell subsets of five monozygotic HLA‐B27+ twins (two concordant and three discordant for AS) and CD8+ T cell repertoires of three healthy HLA‐B27+ individuals were characterized by TCR β‐chain (TCRB) CDR3 size spectratyping. Selected TCRB‐CDR3 spectra were further analysed by BJ‐segment analysis and TCRB‐CDR3 from expanded T cell clones were sequenced. In an analysis of all data (519/598 possible TCRB‐CDR3 spectra), AS was associated with increased T cell oligoclonality in both CD8+ (P = 0·0001) and CD4+ (P = 0·033) T cell subsets. This was also evident when data were compared between individual twins. Nucleotide sequence analysis of expanded CD8+ or CD4+ T cell clones did not show selection for particular TCRB‐CDR3 amino acid sequence motifs but displayed sequence homologies with published sequences from intra‐epithelial lymphocytes or synovial T cells from rheumatoid arthritis patients. Together, these results provide support for the hypothesis that responses to T cell‐stimulating exogenous or endogenous antigens are involved in the induction and/or maintenance of AS.

Thymoma and Pure Red Cell Aplasia in a Patient with Systemic Lupus Erythematosus

We present the case of a female patient with a diagnosis of systemic lupus erythematosus (SLE) at the age of 54 years. At the age of 63 years, she suffered from malignant thymoma and 3 years after removal of the thymoma a diagnosis of pure red cell aplasia (PRCA) was established. This is, to our knowledge, the first report of the occurrence of SLE, thymoma and PRCA in the same patient. The case is discussed with regard to the already known associations between these diseases.