Stephan Strobel

Oral Tolerance, Systemic Immunoregulation, and Autoimmunity

Abstract: Convincing clinical and experimental evidence suggests that the disturbance of important immunoregulatory and suppressive immunological events induced after oral (mucosal) antigen exposure (oral tolerance) may lead to allergic and autoimmune diseases. Within a variety of factors, age of the host and timing of antigen (food) administration are important characteristics in the development of food allergic disease. Induction of tolerance is seen as a Th2 skewed response, which on one side may prevent harmful mucosal immune reactions but on the other side may contribute to adverse responses in the susceptible individual. The primary mechanisms by which tolerance may be mediated include deletion, anergy, suppression, “ignorance,” and apoptosis. Cell‐mediated delayed hypersensitivity reactions (Th1), which are implicated in the development of autoimmune and gastrointestinal diseases, are particularly well suppressed. Regulatory events after mucosal exposure of antigen are not well characterized and remain controversial. The balance between tolerance (suppression) and sensitization (priming) is dependent on several factors, such as: (a) genetic background, (b) nature and dose of antigen, (c) frequency of administration, (d) age at first antigen exposure, (e) immunological status of the host, (f) antigen transmission via breast milk, and others. Overall there is evidence in rodents that multiple low‐dose feeds are more likely to induce regulatory cytokines (e.g., TGF‐β, IL‐10, IL‐4) in part secreted by CD4+CD25+ T regulatory cells. Despite the powerful suppressive effects of oral autoantigen exposure observed in experimental models of autoimmune diseases (including bystander suppression), their translation into clinical trials of autoimmune diseases has not yet yielded the expected beneficial results.

Neutrophil activation in paediatric extracorporeal circuits: effect of circulation and temperature variation

OBJECTIVE Upregulation of neutrophil adhesion molecules (CD11b and L-selectin) and release of a modulating cytokine (IL8) have been reported in vivo and in vitro in adult cardiopulmonary bypass. The aim of this study was to determine whether paediatric bypass preparations have similar influences and whether neutrophil-endothelium interactions are required for IL8 release. METHODS In vitro paediatric cardiopulmonary bypass circuits (n = 15) were constructed (identical to those used clinically), as well as static loops (n = 15) using donor blood. The effects of circulation and temperature (17 degrees C, 25 degrees C, 37 degrees C) on the initiation of acute inflammation were examined. Cellular expressions of neutrophil adhesion molecules CD11b and L-selectin were assayed by immunofluorescence technique, and serum IL8, IL6, TNF-alpha, leucocyte elastase, and terminal complement complex were measured by ELISA. RESULTS In all experiments, an immediate increase in CD11b expression occurred [median values, in relative fluorescence units: 64.9 (range 45.3-212.9) at rest; 365.2 (205-835.4) at 10 min; P < 0.001], along with a decrease in L-selectin expression [153.5 (115.5-220.7) at rest; 42 (12-134) at 10 min; P < 0.01]. Serum concentrations of the following increased gradually and were higher in circulation than in static loops: IL8 [1500 (500-2500) pg.ml-1 in circuit v 600 (180-1500) pg.ml-1 in loop, P < 0.001]; TNF-alpha P < 0.05]; and terminal complement complex [25.9 (6.8-120) v 4.7 (0-21.6) AU.ml-1, P < 0.01]. Cooling decreased and rewarming increased upregulation of CD11b and downregulation of L-selectin and release of IL8. IL6 was undetectable. CONCLUSIONS In the absence of endothelium, in vitro paediatric cardiopulmonary bypass causes profound acute inflammatory changes in donor blood with release of IL8. These changes were greater than in adult cardiopulmonary bypass. Temperature variation and circulation modulate the responses.

Effect of heparin anticoagulation on neutrophil adhesion molecules and release of IL8: C3 is not essential

OBJECTIVE To examine the role of heparin in modulating neutrophil activation and release of cytokine. BACKGROUND Up-regulation of CD11b, down-regulation of L-selectin on neutrophil cell surface and release of IL8 occur in response to extracorporeal circulation (ECC) and were proposed to cause leakage of the capillaries in patients. DESIGN In a series of experiments, we examined the effect of heparin (4 U/ml) comparing it with ethylenediamine tetra-acetate (EDTA, 1.5 mg/ml) and citrate mixture (100 microliters/ml), heparin dose-response, IL8 (human recombinant IL8) dose-response and protamine (80 micrograms/ml) neutralisation of heparin (4 U/ml) using donor blood (total of 38). The role of complement component type 3 (C3) was tested. Neutrophils from a patient with complete C3 deficiency were stimulated by using heparin and cobra venom factor (10 micrograms/ml) and compared with controls (n = 5). CD11b and L-selectin expressions were assayed immediately and serially up to 120 min using immune fluorescence and flow cytometry. Serum concentrations of IL8 were determined by using enzyme-linked immunosorbent assay. RESULTS The medians of up-regulation of CD11b were 540.2 (range 235.2-653.3) for heparin vs. 186.5 (55.7-207.1) for EDTA and 192.5 (69.2-263.8) for citrate mixture, P < 0.01. The medians of down-regulation of L-selectin were 79 (32-192) for heparin vs. 18.4 (0-188) for EDTA and 36.2 (7.4-135) for citrate mixture, P < 0.05. Up-regulation of CD11b, down-regulation of L-s and release of IL8 were inversely related to heparin concentration (r = 0.87, P < 0.05). Serum concentration of IL8 had a direct relationship to the changes in CD11b and L-selectin expression (r = 0.92). Heparin-protamine complex was less stimulant to expression of CD11b and L-selectin than heparin or protamine (P < 0.05). In blood samples from C3-deficient patients, heparin and cobra venom factor caused up-regulation of CD11b and down-regulation of L-selectin similar to that of controls (P > 0.05). CONCLUSIONS Heparin stimulates up-regulation of neutrophil adhesion molecules CD11b, down-regulation of L-selectin and release of IL8. These effects are inversely related to heparin concentration and are independent of C3 activation. IL8 has a direct relationship to activation of neutrophil adhesion molecules. Increasing heparin dosage reduces neutrophil activation and may reduce the morbidity of patients.

Cardiopulmonary bypass tubes and prime solutions stimulate neutrophil adhesion molecules.

OBJECTIVE To evaluate effects of the material of the cardiopulmonary bypass (CPB) tubes (polyvinyl chloride, PVC) and prime solutions on expression of neutrophil adhesion molecule CD11b and L-selectin. METHODS We carried out a series of experiments using donor blood from 30 healthy adult human volunteers. In all experiments, neutrophil cell surface expressions of CD11b and L-selectin were assayed immediately and serially up to 2 hours, using immune-fluorescence techniques and flow cytometry. Study 1: Effects of PVC were compared with glass and polystyrene (n = 5). Study 2: Blood was mixed with Plasma-lyte (Pl) (prime solution), Hartman solutions, albumin or not altered (control), n = 5. Study 3: The effects of changing pH of the Pl (control, neutralised and acidic solution, n = 5) were examined. Study 4: Haemodilution (undiluted, 1:1, 1:2, and 1:3, vol/vol, prime to blood, n = 5) was carried out using Pl and the subsequent changes in expressions of the adhesion molecules were analysed. Study 5: The combined effect of PVC and Pl was assessed (n = 5). Study 6: We evaluated the effect of increasing plasma water by adding sterile water to whole blood and compared it with control (n = 5). RESULTS Study 1: PVC, similar to glass, caused more up-regulation of CD11b and down-regulation of L-selectin than polystyrene (238 and 162% vs. 68 increase of CD11b, P < 0.001; 89 and 95% vs. 16% decrease of L-selectin, P < 0.001). Study 2: Pl and Hartman solutions caused more up-regulation of CD11b and down-regulation of L-selectin compared to albumin and control (166 and 188% vs. 26 and 44% increase of CD11b, P < 0.01; 19 and 26% vs. 10 and 6% decrease of L-selectin, P < 0.01, respectively). Study 3: Haemodilution had no effect on these molecules. Study 4: The mean of the difference between the acidic and neutral solution was 208% increase of CD11b and 30% decrease of L-selectin, P < 0.05. Study 5: The combined effect of mixing blood with Pl and exposure to PVC caused marked up-regulation of CD11b (336% increase, P < 0.01) and down-regulation of L-selectin (78% decrease, P < 0.05). Study 6: Water for injection caused marked up-regulation of CD1 1b and down-regulation of L-selectin. CONCLUSIONS Mixing blood with acidic prime solution and/or exposing it to PVC tubes causes up-regulation of neutrophil adhesion molecule CD11b and down-regulation of L-selectin. Neutralisation of the prime solution reduces the extent of neutrophil activation, whereas haemodilution has no effect. Increasing plasma water is stimulating to the neutrophil. Modulation of prime solutions and the material of CPB tubes may reduce neutrophil activation which may reduce patient morbidity.

Gastrointestinal allergy: clinical symptoms and immunological mechanisms.

The gastrointestinal tract (GIT) and its associated lymphoid organs (gut-associated lymphoid tissues: GALT) is the largest organ of the immune system. This is an evolutionary response to the huge and constant burden of both harmless and dangerous pathogens and antigens that are encountered daily by the GIT. The mechanisms of dynamic immunological control are complex. Perturbation can result in severe or fatal infection by normally tolerated organisms (such as Giardia lamblia or Cryptosporidium species) or in development of intolerance of antigens – including food antigens – that were previously tolerated. The role of neuroendocrine control of GIT immune responses is an underdeveloped area of scientific enquiry. Other areas such as IgE associated food allergy and coeliac disease have long been fruitful areas of research and of huge benefits for affected subjects. This short review aims to outline some of the symptom complexes and the mechanisms thought to underlie some of the common and more unusual gastrointestinal presentations of intolerance and allergic diseases. The aetiology and symptomatology of adverse reactions affecting the gastrointestinal tract are variable. The mechanisms involved and the site of the reaction influence the time taken for symptoms and clinical signs to develop after eating the food product. Any part of the gastrointestinal tract can be involved. The features may occur alone or together as part of a syndrome.

V. Mechanisms of tolerance and sensitization in the intestine and other organs of the body

The intestinal tract is the major immunological organ of the human body.