Platelet-Derived Interleukin-1 Induces Cytokine Production, but not Proliferation of Human Vascular Smooth Muscle Cells by Harald Loppnow, Rosita Bil, Stephan Hirt, Uwe Schönbeck, Mona Herzberg, Karl Werdan, Ernst Theodor Rietschel, Ernst Brandt, and Hans-Dieter Flad Blood Volume 91(1):134-141 January 1, 1998 ©1998 by American Society of Hematology

(A) Platelets stimulate the IL-6 production of SMC in a cell number dependent fashion. (A) Platelets stimulate the IL-6 production of SMC in a cell number dependent fashion. Human vascular SMC were incubated in 24-well plates until they reached confluency. The cultures were washed (RPMI 1640) and medium (squares) or different numbers of isolated platelets (Pla; circles) added. After centrifugation of plates (500g, 10 minutes), medium (open symbols), or medium containing thrombin (1 U/mL; closed symbols) was added. Supernatants were harvested after 24 hours and centrifuged (1,500g, 5 minutes). IL-6 activity in supernatants was measured in 7TD1-assay. Similar results were obtained in 5 experiments. (B) The activation of platelets with thrombin is specific. In a separate experiment SMC were incubated in 24-well plates until they reached confluency, washed, and medium (▪) or medium containing hirudin (20 U/mL; ▨) was added. Subsequently platelets at a ratio of 1,000 platelets per SMC and finally, medium (None) or medium containing thrombin (Thrombin; 1 U/mL) was added. After the incubation (24 hours) supernatants were harvested and IL-6 was measured in 7TD1-assay. Two additional experiments showed similar results. Harald Loppnow et al. Blood 1998;91:134-141 ©1998 by American Society of Hematology

Platelets induce IL-8 production of SMC Platelets induce IL-8 production of SMC. SMC were incubated in 24-well plates until they reached confluency. Platelets induce IL-8 production of SMC. SMC were incubated in 24-well plates until they reached confluency. The cultures were washed and the SMC prestimulated with (+) or without (−) recombinant TNF-α (TNF, 50 ng/mL). Untreated (−) platelets or platelets in the presence of thrombin (+; 1 U/mL) were added at a ratio of 1,000 platelets per SMC. The supernatants were harvested after 24 hours, run on a reducing SDS-PAGE, and blotted to Immobilon P. A rabbit polyclonal antibody (IL-854-72; 1:1,000) was added (1 hour) and the blot stained with peroxidase-conjugated goat-anti-rabbit (1 hour) and subsequent addition of DAB. Similar results were obtained in three experiments. Harald Loppnow et al. Blood 1998;91:134-141 ©1998 by American Society of Hematology

Platelets induce proliferation of SMC Platelets induce proliferation of SMC. SMC were cultured in 24-well culture plates until they reached confluency, washed with serum-free medium, and incubated for 24 hours in serum-free medium containing insulin and transferrin (IT-medium). Platelets induce proliferation of SMC. SMC were cultured in 24-well culture plates until they reached confluency, washed with serum-free medium, and incubated for 24 hours in serum-free medium containing insulin and transferrin (IT-medium). The cultures were then washed, platelets at the denoted ratios added, and the cocultures incubated with or without the respective stimuli (72 hours). Subsequently radiolabeled thymidine was added for 24 hours and thymidine incorporation of cells measured in a scintillation counter (Proliferation). For IL-6 measurement parallel cultures were washed and incubated with the stimuli for 24 hours (IL-6). Two additional experiments showed comparable results. Harald Loppnow et al. Blood 1998;91:134-141 ©1998 by American Society of Hematology

(A) TNF prestimulation of SMC enhances the response to platelets. (A) TNF prestimulation of SMC enhances the response to platelets. SMC were incubated in 24-well plates until they reached confluency, washed, and medium (None) or recombinant TNF (50 ng/mL) was added. After 4 hours SMC were washed and 1,000 platelets per SMC were added. Plates were centrifuged (500g, 10 minutes), and medium lacking thrombin (Medium) or medium containing thrombin (1 U/mL; Thrombin) was added for a further 24 hours. IL-6 activity in supernatants was measured in 7TD1-assay. Four similar experiments were performed. (B) Prestimulation of SMC with LPS, TNF, or IL-1. In a separate experiment SMC were cultured and prestimulated for 4 hours with LPS (1 μg/mL), TNF-α (TNF, 50 ng/mL), or IL-1α (IL-1, 10 ng/mL) as described in (panel A) and thrombin-activated platelets were added. The IL-6 production of SMC not prestimulated but incubated with thrombin-activated platelets (None) was determined 0% and the maximal IL-6 production after IL-1 prestimulation determined 100%. Two similar experiments were performed. Harald Loppnow et al. Blood 1998;91:134-141 ©1998 by American Society of Hematology

Platelets contain IL-1 mRNA. Platelets contain IL-1 mRNA. The total mRNA of platelets or leukocytes was isolated. These preparations were incubated with (+) or without (−) reverse transcriptase for 1 hour and PCR was performed (35 cycles) for IL-1α, IL-1β, and IL-8 receptor type I (IL-8-R). Samples were run on a 1.3% agarose gel and visualized by UV-transillumination. The expected size of the PCR products are 810, 816, and 589 bp, respectively. A 100-bp ladder was included as marker. Three experiments showed similar results. Harald Loppnow et al. Blood 1998;91:134-141 ©1998 by American Society of Hematology

Thrombin and IL-1 synergistically induce IL-6 production of SMC Thrombin and IL-1 synergistically induce IL-6 production of SMC. SMC were incubated in 24-well plates until they reached confluency, washed and stimulated separately (A) with IL-1α (1, 10 or 100 pg/mL) or thrombin (Thr; 0.1, 1, 10 U/mL), or with combination... Thrombin and IL-1 synergistically induce IL-6 production of SMC. SMC were incubated in 24-well plates until they reached confluency, washed and stimulated separately (A) with IL-1α (1, 10 or 100 pg/mL) or thrombin (Thr; 0.1, 1, 10 U/mL), or with combinations (B) of IL-1α and thrombin. IL-6 activities detected in supernatants of SMC costimulated with combinations of IL-1α and thrombin are shown in (B). For estimation of the synergistic effect IL-6 activities in supernatants of SMC stimulated separately with IL-1α or thrombin are added up and presented in (A). IL-6 activity in supernatants was measured in 7TD1-assay. Four independent experiments showed similar results. The asterisks indicate costimulation cocultures expressing a synergistic effect. Harald Loppnow et al. Blood 1998;91:134-141 ©1998 by American Society of Hematology