Veit Krenn

Expression of the integrin αEβ7 identifies unique subsets of CD25+ as well as CD25− regulatory T cells

Regulatory CD25+CD4+ T cells are considered as important players in T cell homeostasis and self-tolerance. Here we report that the integrin αEβ7, which recognizes epithelial cadherin, identifies the most potent subpopulation of regulatory CD25+ T cells. Strikingly, CD25-negative αE+CD4+ T cells displayed regulatory activity. Both αE+ subsets, CD25+ and CD25−, express CTLA-4, suppress T cell proliferation in vitro, and protect mice from colitis in the severe combined immunodeficient model (SCID) in vivo. Whereas αE+CD25+ T cells produce almost no cytokines, αE+CD25− T cells represent a unique subset in which high IL-2, IFN-γ and T helper 2-cytokine production is linked with suppressive function. Thus, the integrin αEβ7 can be regarded as a novel marker for subsets of highly potent, functionally distinct regulatory T cells specialized for crosstalk with epithelial environments.

Synovitis score: discrimination between chronic low-grade and high-grade synovitis

Aims : To standardize the histopathological assessment of synovial membrane specimens in order to contribute to the diagnostics of rheumatic and non‐rheumatic joint diseases.

Inflamed kidneys of NZB / W mice are a major site for the homeostasis of plasma cells.

(NZB × NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody‐secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full‐blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self‐antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA‐specific ASC are found initially in the spleen. Weeks later, OVA‐specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220+ / PNA+) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasisof plasma cells, irrespective of their specificity.

Grading of Chronic Synovitis — A Histopathological Grading System for Molecular and Diagnostic Pathology

The following is a proposition for a simple histopathological classification system to measure inflammation in synovial tissue. This synovitis-score is employed in conventionally stained routine sections, and is applicable to every kind of synovitis, irrespective of etiology and including the following relevant morphological alterations. First: hyperplasia/enlargement of synovial lining cell layer. Second: activation of resident cells/synovial stroma. Third: inflammatory infiltration. All defined histopathological qualities are graded from absent (0), slight (1) and moderate (2) to strong (3), with summaries ranging from 0 to 9. 0 to 1 corresponds to no synovitis (inflammatory grade = 0), 2 to 3 to a slight synovitis (inflammatory grade 1), 4 to 6 to a moderate synovitis (inflammatory grade 2), and 7 to 9 to a strong synovitis (inflammatory grade 3). Using this score, we analyzed 308 random specimens of synovial tissue from degenerative (osteoarthritis (OA)) and inflammatory joint diseases - reactive arthritis (ReA), psoriasis arthritis (PA) and rheumatoid arthritis (RA) - as well as synovial tissue from healthy individuals. The mean grade given to synovitis of RA turned out to be significantly higher than the mean grade of OA (p < 0.0005) and of healthy controls (p < 0.0005). On the contrary, no significant differences could be found between the mean grades of synovitis scores from patients with RA and those with PA and ReA. Another comparison between RA-synovitis types I and II according to the Stiehl classification resulted in type I (p < 0.0005), showing significantly higher values of inflammatory infiltration, and type II (p = 0.037), showing significantly higher values of stroma activation. Since in OA, synovitis is regarded as a result of degenerative cartilage destruction whereas in inflammatory joint diseases (RA, PA, ReA), synovitis is regarded to be the cause of cartilage destruction, it can be concluded that scores with considerable high values indicate the pathogenetic potential of synovitis and that the inflammatory score may be helpful in estimating the destructive potential of synovitis at the same time. Furthermore, the comparison of the score data with the Stiehl RA-synovitis types shows that the score enables us to discriminate the morphological peculiarities of the synovitis types. In experimental pathology, it could provide standardized information on molecular synovial tissue analyses where a correlation of molecular with morphological data is essential. In diagnostic pathology, this synovitis score (in combination with other typing systems) could provide basic and standardized information concerning the degree of inflammatory alterations in synovial tissue.

Immunization with Glucose-6-Phosphate Isomerase Induces T Cell-Dependent Peripheral Polyarthritis in Genetically Unaltered Mice

Rheumatoid arthritis is a chronic inflammatory disease primarily affecting the joints. The search for arthritogenic autoantigens that trigger autoimmune responses in rheumatoid arthritis has largely focused on cartilage- or joint-specific Ags. In this study, we show that immunization with the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI) induces severe peripheral symmetric polyarthritis in normal mice. In genetically unaltered mice, T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4+ cells. In contrast, Abs and FcγR+ effector cells are necessary but not sufficient for G6PI-induced arthritis in genetically unaltered mice. Thus, the complex pathogenesis of G6PI-induced arthritis in normal mice differs strongly from the spontaneously occurring arthritis in the transgenic K/B × N model where Abs against G6PI alone suffice to induce the disease. G6PI-induced arthritis demonstrates for the first time the induction of organ-specific disease by systemic autoimmunity in genetically unaltered mice. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.

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.

Revised histopathological consensus classification of joint implant related pathology

This extended classification of joint implant related pathology is a practical histopathologic classification based on defined morphological criteria covering the complete spectrum of pathohistologic changes in periprosthetic tissues. These changes may occur as a consequence of endoprosthetic replacement of large joints and may lead to a reduction in the prosthesis survival rate. We describe the established consensus classification of the periprosthetic membrane, in which aseptic and septic prosthetic loosening can be subdivided into four histological types, as well as histopathological criteria for additional significant pathologies including endoprosthetic-associated arthrofibrosis, particle-induced immunological, inflammatory and toxic mechanisms (adverse reactions), and bone tissue pathologies. These characteristic tissue alterations and their relationships are summarized in the extended classification. Since particle heterogeneity in periprosthetic tissue is high and particle identification is a necessary part of diagnosis, the identification of different types of particles is described in the histopathological particle algorithm. The morphological qualities of prosthetic material particles and the demarcation between abrasion and non-abrasion endogenous particles are also summarized. This feasible classification which is based on low cost standard tissue processing and examination and on well-defined diagnostic criteria is a solid platform for the histological diagnosis of implant associated pathologies providing a stable and reproducible tool for the surgical pathologist. Since this classification is suitable for standardized histopathological diagnostics, it might also provide a useful data set for joint arthroplasty registers, particularly for registers based on so-called routine data.

Modified immunohistological staining allows detection of Ziehl-Neelsen-negative Mycobacterium tuberculosis organisms and their precise localization in human tissue

The diagnosis of mycobacterial infection depends on the Ziehl–Neelsen (ZN) stain, which detects mycobacteria because of their characteristic acid‐fast cell wall composition and structure. The histological diagnosis of tuberculosis (TB) comprises various aspects: (1) sensitive detection of mycobacteria; (2) precise localization of mycobacteria in the context of granulomatous lesions; (3) ‘staging’ of disease according to mycobacterial spread and granulomatous tissue integrity. Thus, detection of minute numbers of acid‐fast bacteria in tissue specimens is critical. The conventional ZN stain fails to identify mycobacteria in numbers less than 104 per ml. Hence many infections evade diagnosis. PCR is highly sensitive, but allows neither localization within tissues nor staging of mycobacterial disease, and positive findings frequently do not correlate with disease. In this study, an anti‐Mycobacterium bovis bacille Calmette–Guérin polyclonal antiserum (pAbBCG) was used to improve immunostaining, which was compared to the ZN stain in histological samples. Screening of tissue samples including lungs, pleural lesions, lymph nodes, bone marrow, and skin for mycobacterial infection revealed that pAbBCG staining detects infected macrophages harbouring intracellular mycobacteria or mycobacterial material as well as free mycobacteria that are present at low abundance and not detected by the ZN stain. The positive pAbBCG staining results were confirmed either by PCR analysis of microdissected stained tissue or by culture from tissue. This immunostaining approach allows precise localization of the pathogen in infected tissue. Copyright

C4d in Acute Rejection After Liver Transplantation—A Valuable Tool in Differential Diagnosis to Hepatitis C Recurrence

Hepatitis C is the most common indication for liver transplantation. Recurrence of HCV is universal leading to graft failure in up to 40% of all patients. The differentiation between acute rejection and recurrent hepatitis C is crucial as rejection treatments are likely to aggravate HCV recurrence. Histological examination of liver biopsy remains the gold standard for diagnosis of acute rejection but has failed in the past to distinguish between acute rejection and recurrent hepatitis C. We have recently reported that C4d as a marker of the activated complement cascade is detectable in hepatic specimen in acute rejection after liver transplantation. In this study, we investigate whether C4d may serve as a specific marker for differential diagnosis in hepatitis C reinfection cases. Immunohistochemical analysis of 97 patients was performed. A total of 67.7% of patients with acute cellular rejection displayed C4d‐positive staining in liver biopsy whereas 11.8% of patients with hepatitis C reinfection tested positive for C4d. In the control group, 6.9% showed C4d positivity. For the first time we were able to clearly demonstrate that humoral components, represented by C4d deposition, play a role in acute cellular rejection after LTX. Consequently C4d may be helpful to distinguish between acute rejection and reinfection after LTX for HCV.

Description of B lymphocytes and plasma cells, complement, and chemokines/receptors in acute liver allograft rejection.

Background. Although antibody mechanisms play a pathogenetic role in liver allograft rejection, no data exist on B lymphocytes, plasma cells, complement, and chemokines in rejected liver tissue. Methods. Liver biopsy specimens from 25 patients with acute allograft rejection (AR) (rejection activity index, RAI score: 1–9) were analyzed by immunohistochemistry (IH) and reverse transcriptase-polymerase chain reaction (RT-PCR) and compared with biopsy specimens taken prior to implantation (PI). The number of CD20+ and CD138+ cells was evaluated, and the presence and abundance of the chemokines macrophage inflammatory protein (MIP)-3&agr;, CXCL9, CXCL10, CXCL11, CXCL12, and their receptors CCR-6, CXCR3, and CXCR4 were examined. Complement depositions were visualized by C4d IH. Results. The numbers of B lymphocytes (P=0.002) and plasma cells (P=0.022) were significantly higher in AR biopsy specimens compared with PI biopsy specimens. MIP-3&agr;+ and CCR-6+ cells were detected in the portal fields of all AR biopsy specimens. IH double staining revealed a colocalization of MIP-3&agr;+/CD20+ cells; C4d deposits could be demonstrated along the portal capillaries. All examined chemokines and receptors could be detected in normal liver tissue and in AR biopsy specimens by RT-PCR and semiquantitative RT-PCR, demonstrating an overexpression of CXCL10 and -11. Conclusions. The significant increase of B lymphocytes and plasma cells during acute rejection, together with the lack of a significant increase of proliferating cells, indicates that the migration of B lymphocytes and plasma cells—promoted by the expression of B-cell activating chemokines/receptors—plays a key role in acute liver rejection. The C4d deposits along the portal capillaries indicate a humorally mediated alloresponse caused by the accumulated B and plasma cells.