Studies on the mechanisms of leukocyte adhesion to cellulose acetate beads: an in vitro model to assess the efficacy of cellulose acetate carrier-based granulocyte and monocyte adsorptive apheresis.

Granulocyte and monocyte adsorptive apheresis (GMA) using a column filled with cellulose acetate (CA) beads (carriers) has been associated with a significant clinical efficacy in patients with rheumatoid arthritis and ulcerative colitis. To obtain further understanding on the mechanisms of disease modification by cellulose acetate-carrier-based GMA, in the present study, we investigated the mechanisms of granulocyte and monocyte adhesion to CA beads following exposure of human peripheral blood to the carriers at 37 degrees C for up to 60 min under controlled conditions. Cellulose acetate beads selectively adsorbed granulocytes, monocytes. CD19+ (B cells) and CD56+ (NK cells) lymphocyte subpopulations. The granulocyte and monocyte adsorption was inhibited by heat-inactivated plasma and EDTA, indicating that the adsorption was plasma protein (immunoglobulin, complement) and calcium dependent. Accordingly, granulocyte and monocyte adsorption was markedly enhanced by coating the carriers with IgG. Similarly, C3b was adsorbed onto the CA beads as a marker of complement activation. The results indicated that IgG and active complement fragments mediated leukocyte adhesion to CA beads via the FcgammaR and/or leukocyte complement receptor like CR3. Additionally, CA beads induced loss of expression of TNF receptors on CD16- granulocytes and CD14+ monocytes, but not on CD3+ lymphocytes In conclusion, CA beads might be an appropriate biomaterial for inducing extracorporeal immunomodulation as a treatment for auto-immune diseases which are associated with pathological leukocyte activity.

Leucocytapheresis with Adacolumn enhances HCV-specific proliferative responses in patients infected with hepatitis C virus genotype 1.

The most important aim in controlling virus infections is to destroy infected cells. Impaired cellular immunity in HIV and HCV infection leads to chronic infection. This study examined the effect of cytapheresis on the subsequent response to interferon/ribavirin treatment in patients infected with HCV. Adacolumn cytapheresis was carried out once a day for 5 consecutive days in patients who relapsed or did not respond to previous peginterferon and ribavirin combination treatment (n = 14: relapsers = 3, non-responders = 11). Peginterferon and ribavirin combination treatment was started after cytapheresis. During combination treatment, the proliferative response of peripheral blood mononuclear cells to HCV proteins (core, NS3, NS4, and NS5), tetanus toxoid, and phytohemagglutinin was measured, and compared to the early virological response. After treatment by leucocytapheresis, the proliferative response of peripheral blood mononuclear cells to HCV-core and tetanus toxoid increased significantly over the baseline (P < 0.05). A marked increase in the phytohemagglutinin response was observed after peginterferon and ribavirin combination treatment was started (P < 0.01 at week 5 and P < 0.005 at week 13). There were, however, no clear changes in the proliferative response to other antigens. Among the 14 patients, 12 (85.7%) achieved an early virological response by week 13 (12 weeks after the start of combination treatment). After treatment, nine patients (64.3%) had a significant proliferative response to HCV core antigen. Among the nine patients, eight patients (88.9%) achieved early virological response. The results indicate that activation of cellular immunity by leucocytapheresis facilitates an early virological response rate in HCV patients. This new therapy may, therefore, become an additional therapeutic measure for HCV.

In vitro and in vivo evaluation of Adacolumn cytapheresis in healthy subjects.

Adacolumn is a medical device for adsorptive cytapheresis. It has been developed for selective adsorption of granulocytes and monocytes from peripheral blood of patients with immune disorders, such as autoimmune diseases and chronic inflammatory diseases. A double blind sham-controlled crossover study design was used in order to evaluate in vivo biological responses of leukocytes as well as biocompatibility during and after Adacolumn cytapheresis in healthy volunteers. In addition, experiments were undertaken to further evaluate leukocyte reactions to Adacolumn carrier (G-1: cellulose diacetate) beads in vitro. Six healthy volunteers, 4 males and 2 females, with a mean age of 26.7 years were randomly assigned to one of the two treatment arms in a crossover fashion. Three subjects received a single Adacolumn treatment, followed by a single sham treatment at an interval of 7 days. The other three subjects received the two treatments in reverse order. All subjects were followed up 7 days after the last treatment. Additionally, in vitro investigations were carried out using blood from the healthy donors to examine the effect of G-1 beads on granulocyte functions. In vitro exposure of human peripheral blood to G-1 beads caused downregulation of L-selectin expression and upregulation of Mac-1 expression on granulocytes, leading to a marked reduction of adhesive capacity of granulocytes to endothelial cells. The exposure also led to decreased granulocyte chemotactic activity to IL-8. The number of granulocytes and monocytes clearly decreased during Adacolumn cytapheresis. Granulocytes showed marked phenotypic changes of L-selectin(Low) and Mac-1(Hi) after passing through Adacolumn in vivo. Expression of TNF-alpha and chemokine receptors was downregulated. In addition, TNF-alpha and IL-1beta producing capacity of peripheral blood leukocytes was decreased after Adacolumn cytapheresis and these changes lasted even one week after the cytapheresis. The level of complement fragments, C3a and C5a, increased, while bradykinin concentration did not change during Adacolumn cytapheresis. Exposure of human peripheral blood to G-1 beads, both in vitro and in vivo, caused a significant reduction of adhesive capacity and proinflammatory cytokine producing capacity of peripheral blood leukocytes. Such changes were not observed after sham apheresis. Despite complement activation, tolerability of Adacolum cytapheresis was not influenced. These findings may at least partly explain the beneficial effect of Adacolumn cytapheresis in the treatment of autoimmune diseases.

Immunomodulatory effects of granulocyte and monocyte adsorption apheresis as a treatment for patients with ulcerative colitis.

Our aim was to understand the mechanism of immunological changes associated with the use of an adsorptive-type extracorporeal device (Adacolumn) that has been developed for selective adsorption of granulocytes and monocytes/macrophages from peripheral blood of patients with active ulcerative colitis. The column is filled with carriers (G-1 beads) that have a diameter of 2 mm and are made of cellulose diacetate. In peripheral blood treated with the G-1 beads or peripheral blood from patients with active ulcerative colitis following granulocyte and monocyte adsorption apheresis, a significant suppression of proinflammatory cytokines (tissue necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-8) production by leukocytes, neutrophil chemotaxis, down-regulation of leukocyte adhesion molecule (L-selectin) and neutrophil adhesion to interleukin-1beta-activated endothelial cells were observed. Furthermore, after granulocyte adsorption therapy, the number of CD10-negative premature granulocytes increased, indicating increased turnover of these cells in the circulation. Our observations suggest that selective granulocyte and monocyte adsorption is associated with modified peripheral blood leukocyte function favorable to patients with ulcerative colitis and possibly other autoimmune disorders which reflect leukocyte hyperactivity.