In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils.

In order to address neutrophil activation during inflammation we assessed the expression of interleukin 1 receptor type 1 (IL-1R1) following in-vivo extravasation. Extravasated neutrophils were collected from 11 healthy study subjects by a skin chamber technique and compared to neutrophils in peripheral blood. Expression of IL-1R1 was assessed by microarray, quantitative polymerase chain reaction (qPCR), Western blot, flow cytometry, enzyme linked immunosorbent assay (ELISA) and immunoelectron microscopy (iEM). IL-1R1 was induced following extravasation, demonstrated by both gene array and qPCR. Western blot demonstrated an increased expression of IL-1R1 in extravasated leucocytes. This was confirmed further in neutrophils by flow cytometry and iEM that also demonstrated an increased intracellular pool of IL-1R1 that could be mobilized by N-formyl-methionine-leucine-phenylalanine (fMLP). Stimulation of peripheral neutrophils with IL-1 resulted in transcription of NFκB and a number of downstream chemokines and the corresponding chemokines were also induced following in-vivo extravasation. The present results demonstrate that IL-1R1 is induced following extravasation and exists on the neutrophil surface, as well as in a mobile intracellular pool. Furthermore, neutrophils express functional IL-1R1 as demonstrated by the induction of chemokines following IL-1 stimulation. The results indicate a potential role for IL-1 in the activation of neutrophils at inflammatory sites.

IL-8 from local subcutaneous wounds regulates CD11b activation.

The cellular and soluble mediators of a dermal inflammation can be studied by the skin chamber technique. The aim of this study was to address the physiological effect of soluble mediators, released into the skin chamber, with special focus on neutrophil CD11b activation. Mediators released at the inflammatory site were studied by Milliplex and enzyme-linked immunosorbent assay (ELISA) and correlated with transmigration and CD11b activation in vivo and in vitro. Transmigration was studied by the skin chamber technique and by the transwell method, and expression of the CBRM1/5 epitope on activated CD11b was analysed by flow cytometry following in vivo and in vitro incubation with chamber fluid or recombinant interleukin-8 (IL-8). Leucocyte in vivo and in vitro transmigration both correlated with the concentrations of IL-1ß, tumour necrosis factor alpha (TNFα) and IL-8 at P < 0.05 (R > 0.7). Furthermore, CD11b was activated, in terms of exposure of the activation epitope, on neutrophils after 30 min of in vitro incubation with chamber fluid and correlated solely with the concentration of IL-8, P < 0.05 (R = 0.72). In vitro incubation with recombinant IL-8 confirmed a concentration-dependent expression of the activation epitope; however, induction of CBRM1/5 by recombinant IL-8 required a concentration that was significantly higher compared with that in chamber fluid. In addition, the CBRM1/5 epitope was analysed on in vivo extravasated neutrophils that displayed a significantly higher expression compared with circulating neutrophils, P = 0.04. We conclude that IL-8 is the major factor regulating the expression of CD11b activation epitope in neutrophils.

Kinetics of the soluble IL-1 receptor type I during treatment with an LCAP filter in patients with inflammatory bowel disease.

Leukocyte apheresis primarily used for treatment of inflammatory diseases such as inflammatory bowel disease (IBD). Beside an effect of the apheresis column, the plastic lines in the apheresis system might also have an effect due to interaction between the plastic surfaces and circulating leukocytes and plasma proteins. We recently reported generation of LL-37 in the plastic lines during leukocyte adsorbing apheresis. This generation might have a positive impact on the immunologic tolerance and therefore be one operational mechanism by which the apheresis treatment executes its effect. In the present study, we report a significant generation of sIL-1RI in the apheresis lines that is initially absorbed by the LCAP device. This finding, together with our previous data on IL-1Ra indicate that important members of the IL-1 family are significantly altered during the LCAP treatment of patients with IBD. Since IL-1 and its antagonists are important for regulation of inflammatory processes in IBD, we speculate that the LCAP related changes in sIL-1RI and IL-1Ra might impact the clinical outcome. These findings have to be taken into consideration when designing new apheresis techniques as well as sham-controlled studies.

In vivo extravasated human monocytes have an altered expression of CD16, HLA-DR, CD86, CD36 and CX(3)CR1.

The phenotypic alterations in monocytes induced by extravasation in vivo are still largely unknown. We addressed the question whether a general phenotype of extravasated monocytes exists and whether this phenotype differs between healthy individuals and statin treated patients with coronary artery disease (CAD). In vivo extravasated monocytes from CAD patients and healthy controls were collected by use of the skin blister method and compared with peripheral circulating monocytes by flow cytometry. The number of CD14(+)CD16(+) monocytes were significantly higher in the skin blister compared with peripheral circulation in both patients (P < 0.001) and controls (P = 0.005). In vivo extravasated monocytes had in comparison with peripheral monocytes a lower expression of CX(3)CR1, a higher expression of HLA-DR, CD86 and CD36 and a higher binding of acetylated low density lipoprotein (acLDL) (significant for all markers). Skin blister fluid from CAD patients, compared with healthy controls, induced a 20% increase in monocyte CD36 expression (P = 0.008) following 18 h of in vitro incubation. The results indicate that the integrated response to the in vivo extravasation process is similar in statin treated stable CAD patients and healthy controls, with respect to phenotypic alterations. Such differences in CAD patients may, however, occur as a response to the inflammatory milieu.

In vivo transmigrated monocytes from patients with stable coronary artery disease have a reduced expression of CD11b.

Coronary artery disease (CAD) is characterized by infiltration of monocyte derived cells in the intima of the vessel wall. We hypothesized that accumulation of these cells is caused partly by an altered monocyte transmigration process in CAD. To gain insight into this issue we applied the skin blister method that allows collection of in vivo transmigrated cells at sites of local inflammation. Nineteen patients with stable CAD and 19 matched controls were enrolled. Markers of inflammation and gradients of chemokines, as well as adhesion molecule expression and up-regulation capacity, were studied. The expression of inflammatory markers, such as C-reactive protein, interleukin (IL)-6, tumour necrosis factor-alpha and IL-10, was similar in patients and controls, indicating that patients were in a stable phase of the disease. Expression of adhesion molecules, CD11b and very late activation antigen-4, on peripheral monocytes did not differ between patients and controls. However, following in vivo transmigration, monocytes in patients with CAD had a significantly reduced expression and mobilization of CD11b. The effect on CD11b could not be reproduced by in vitro stimulation with blister fluid, representing a local inflammatory milieu, or in an in vitro system of transmigration. These findings point towards differences in monocyte CD11b expression and availability at an inflammatory site between patients with CAD and healthy controls.