Conny Höflich

Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by Sympathetic Activation Reversal by Poststroke T Helper Cell Type 1–like Immunostimulation

Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the susceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-γ–deficient mice, or administration of IFN-γ at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN-γ response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, administration of the β-adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke.

Immunopathogenesis of psoriasis

Abstract:u2002 Psoriasis is a chronic skin disease that affects about 1.5% of the Caucasian population and is characterized by typical macroscopic and microscopic skin alterations. Psoriatic lesions are sharply demarcated, red and slightly raised lesions with silver‐whitish scales. The microscopic alterations of psoriatic plaques include an infiltration of immune cells in the dermis and epidermis, a dilatation and an increase in the number of blood vessels in the upper dermis, and a massively thickened epidermis with atypical keratinocyte differentiation. It is considered a fact that the immune system plays an important role in the pathogenesis of psoriasis. Since the early 1990s, it has been assumed that T1 cells play the dominant role in the initiation and maintenance of psoriasis. However, the profound success of anti‐tumor necrosis factor‐α therapy, when compared with T‐cell depletion therapies, should provoke us to critically re‐evaluate the current hypothesis for psoriasis pathogenesis. Recently made discoveries regarding other T‐cell populations such as Th17 and regulatory T cells, dendritic cells, macrophages, the keratinocyte signal transduction and novel cytokines including interleukin (IL)‐22, IL‐23 and IL‐20, let us postulate that the pathogenesis of psoriasis consists of distinct subsequent stages, in each of them different cell types playing a dominant role. Our model helps to explain the varied effectiveness of the currently tested immune modulating therapies and may enable the prediction of the success of future therapies.

The enigma of CD57+CD28– T cell expansion—anergy or activation?

Expansion of a CD57+CD8+u2003T lymphocyte subset has been reported in HIV and human cytomegalovirus (HCMV) infection. Almost all of these T cells lack CD28 expression. While CD28– cells are often associated with anergy, some authors believe their expansion in HIV infection precipitates immunodeficiency. We studied 15 randomly chosen patients with immune activation and observed that CD57+CD28– T cell expansion may occur in various conditions and to the same degree as in HIV infection without resulting in immunodeficiency. Triple colour flow cytometry also revealed that the CD57 and CD28 antigens are coexpressed in only 3% of CD8+u2003T cells, irrespective of the underlying condition, so that almost all CD57+CD8+ cells are always CD28–. Analysis of Fas (CD95) expression with respect to CD28 expression on CD4+ and CD8+u2003T cells from 10 additional patients indicated no increased commitment to apoptosis in CD28– T cells. Semiquantitative polymerase chain reaction (PCR) comparing CD28+ and CD28–CD8+u2003T cells with respect to cytokine gene expression (tumour necrosis factor‐alpha (TNF‐α), interferon‐gamma (IFN‐γ), IL‐1β) in five renal transplant patients with expansion of the CD57+ subset detected no cytokine gene expression deficit in CD28– T cells. A direct association of increased proportions of CD57+CD28–CD8+u2003T cells with immunodeficiency/anergy is disputed.

The evaluation of psoriasis therapy with biologics leads to a revision of the current view of the pathogenesis of this disorder

Psoriasis is a common chronic, recurring skin disease that is characterised by typical macroscopic and microscopic skin alterations. It is widely accepted that the immune system plays an important role in the pathogenesis of this disorder. Since the early 1990s, the dominant role of a subpopulation of T cells, so-called T1 cells, and their prominent cytokine IFN-γ has been assumed in the pathogenesis of psoriasis. Surprisingly, the comparison of the therapeutic success of treatments with recombinant proteins directed against defined immunological structures shows that those that directly affect T cells (alefacept, efalizumab, Hu-max-CD4, OKTcdr4a) were clearly less effective than those targeting TNF-α (etanercept, adalimumab, infliximab). For this reason, the authors critically re-evaluated the view of psoriasis pathogenesis and postulate that in the majority of patients the T1 cells do not play a dominant role in the clinical, visible stage of this disease.

Reduced monocyte CD86 expression in postinflammatory immunodeficiency.

Objective:Major surgery, polytrauma, stroke, and pancreatitis frequently lead to a compensatory anti-inflammatory response syndrome that often predisposes patients to lethal infections. This temporary postinflammatory immunodeficiency is characterized by altered function of blood monocytes. These cells show strongly reduced inflammatory and antigen-presentation capacity. Diminished monocyte expression of the major histocompatibility complex class II molecule human leukocyte antigen (HLA)-DR is a well-established diagnostic marker of this immunodeficiency. To further characterize the monocytic cells in this clinical state, we analyzed their expression of CD86, the most important co-stimulatory molecule. Design:Analysis of blood samples that entered the clinical immunologic diagnostics and of cells from an in vitro model of postinflammatory immunodeficiency. Setting:University laboratory. Subjects:Healthy donors and intensive care unit (ICU) patients at the university hospital. Interventions:None. Measurements and Main Results:The expression of HLA-DR on monocytes and of CD86 and CD80 on monocytes and B cells was analyzed by flow cytometry. Messenger RNA expression of CD86 was analyzed in isolated monocytes by real-time polymerase chain reaction on reverse transcribed. The normal range of monocyte CD86 expression in healthy subjects was established to be from 2128 to 5102 surface molecules per cell and was independent of age, gender, and leukocyte and monocyte count. The CD86 expression on monocytes in ICU patients correlated with HLA-DR expression. Approximately 40% of the ICU patients with long-term reduced monocyte HLA-DR expression had a long-term reduction of CD86 expression. Patients in whom the expression of both molecules was diminished had an unfavorable prognosis. The diminished number of CD86 surface molecules on monocytes was associated with reduced CD86 messenger RNA levels in these cells. The expression of CD86 in B cells was not diminished in immunodeficient patients. The expression of CD80 in both monocytes and B-cells was minimal in healthy donors and not clearly changed in patients. Conclusions:The monocyte CD86 expression may be a helpful diagnostic variable in ICU patients.

A Simple Assay to Measure Phagocytosis of Live Bacteria

BACKGROUNDnThe phagocytosis of pathogens is essential for fighting infections. No assay is available, however, to measure both engulfment and degradation of bacteria under conditions similar to those in vivo. We sought to develop a flow cytometric assay to measure the engulfment and degradation of live bacteria by human blood monocytes and granulocytes.nnnMETHODSnWe generated enhanced green fluorescent protein (EGFP)-expressing Eschericha coli by transforming E. coli with the plasmid vector pEGFP. We used these bacteria in a flow cytometric assay to measure both engulfment and degradation of living bacteria by monocytes and granulocytes in human whole blood from fresh, heparinized venous blood samples. To determine whether the test detected differences between healthy individuals and patients with secondary immunodeficiencies, we compared the phagocytosis of monocytes and granulocytes measured in blood samples from immunosuppressed kidney transplantation patients and from patients with postoperative sepsis in immunoparalysis with phagocytosis measured in samples from age-matched healthy individuals.nnnRESULTSnIn samples from healthy individuals, we found that in both monocytes and granulocytes bacterial degradation was negatively correlated with the age of the sample donor. Furthermore, we detected decreased bacterial engulfment in granulocytes from septic patients and decreased bacterial degradation in monocytes from immunosuppressed kidney transplantation patients.nnnCONCLUSIONSnThis flow cytometric assay measures the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. By means of this assay we detected significant differences between healthy controls and patients with secondary immunodeficiencies that may contribute to the increased incidence of infection complications seen in these patients.

Differential expression and function of α-mannosidase I in stimulated naive and memory CD4+ T cells.

N-linked protein glycosylation represents an important cellular process for modifying protein properties. It resembles a cascade of various enzymatic reactions, in which class I &agr;-mannosidases play a central role. We and others have recently shown that N-glycosylation plays a major role for immune functions. We now analyzed the expression and function of &agr;-mannosidase I in CD4+ naive and memory T cells studying human and murine T cells. Alpha-mannosidase I function was altered by (i) treatment with Kifunensine, a specific inhibitor class I &agr;-mannosidases, (ii) synthetic inhibitory RNA, and (iii) overexpression by retroviral gene transfer. T-cell activation was evaluated by CD69 expression, cytokine production and proliferation. Our results demonstrate (i) that &agr;-mannosidase I transcription is transiently downregulated after T-cell activation with either polyclonal anti-CD3/CD28 antibodies or allogeneic CD19+ B cells, and (ii) that &agr;-mannosidase I exerts an inhibitory effect on T-cell activation. It is interesting to note that the inhibitory effect was restricted to naive CD4+ T cells in both systems, human T cells and murine transgenic CD4+OT-II cells, whereas human memory T cells and primed CD4+OT-II cells remained unaffected. Alpha-mannosidase I inhibition reduced the activation threshold for naive but not already primed CD4+OT-II cells as the cells were able to respond to lower ovalbumin peptide concentrations and increased the rejection potential of alloreactive T cells in vivo. Thus, complex N-glycans generated by enzymes such as &agr;-mannosidase I inhibit the activation of naive T cells. These findings could be used to improve the ex vivo priming of naive T cells for adaptive T-cell therapies.

Long‐term interleukin‐10 presence induces the development of a novel, monocyte‐derived cell type

Interleukin (IL)‐10 is one of the most crucial immunoregulatory cytokines. Its short‐term effects have been analysed extensively, but little is known about its long‐term effects. This is of considerable importance, as high systemic IL‐10 levels are present for long periods in patients with persistent viral infections, certain cancers and in critical care patients. Our study investigated the effects of the long‐term presence of IL‐10 on human peripheral blood monocytes. In vitro, IL‐10 treatment of these cells for 7u2003days induced the development of a novel cell type characterized by unique phenotypical and functional characteristics. These cells showed high HLA‐DR expression and low expression of CD86 and other co‐stimulatory molecules on their surface. The mRNA levels of both HLA‐DR and CD86 were high, but no intracellular accumulation of CD86 protein was observed. With respect to its function, these cells showed strongly diminished tumour necrosis factor‐α production following lipopolysaccharide stimulation, strongly diminished allogenic CD4+ T cell stimulatory capacity, and even induced a hyporesponsive state in CD4+ T cells. The phenotype remained stable despite the removal of IL‐10. In vivo, we found monocytic cells from patients exhibiting this phenotype after long‐term IL‐10 exposure. These results complement our knowledge further about the biological effects of IL‐10 and may provide an explanation for the sustained immunodeficiency in cases of the persistent presence of systemic IL‐10.

Rapid Whole Blood Flow Cytometric Test to Detect ICOS Deficiency in Patients with Common Variable Immunodeficiency

Background/Aims: Common variable immunodeficiency (CVID) is the most common primary immunodeficiency. With respect to underlying defects it comprises a heterogeneous group of deficiencies. For some patients, distinct phenotypical abnormalities have been described, e.g. partial CD40L deficiency or complete ICOS deficiency. For the diagnosis of CD40L deficiency, a rapid whole blood flow cytometric method has been described several years ago. We aimed to determine if the same method can be used to diagnose ICOS deficiency. Methods: Whole blood from 8 healthy volunteers was stimulated for 4 and 20 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Induction of ICOS expression was analyzed on CD8–CD3+ lymphocytes using three-color flow cytometry. Blood from a patient with diagnosed ICOS deficiency was also analyzed. Results: Whole-blood stimulation with PMA and ionomycin for 20 h resulted in a significant induction of ICOS expression on CD8–CD3+ lymphocytes in healthy volunteers. Four-hour incubation also demonstrated ICOS upregulation but to a much lower extent. In CD8–CD3+ lymphocytes from an ICOS-deficient patient, no ICOS expression could be induced following 20 h of stimulation. Conclusion: ICOS expression can be analyzed using a rapid whole blood flow cytometric test.

Immunomodulatory Therapies: Challenges of Individualized Therapy Strategies

Individualized therapy strategies involve strategies that allow treatment to be guided by patient-specific conditions. For this, robust biomarkers are needed. Examples of biomarker-guided therapies already in use are the treatment of insulin-dependent diabetes (biomarker: blood glucose level) or the treatment of hypertension (biomarker: blood pressure). By contrast, most immunomodulatory therapies are given according to the patients body weight or the patients drug blood level rather than according to biomarkers indicating the patients state of the immune system. Herein we report on new biomarkerguided studies in the immunosuppressive treatment of transplant patients and patients with autoimmune disease and we discuss its benefits and pitfalls.