Fibroblast IL-1-P
IL-l-a and IL-l-p are closely related moieties that are frequently produced simultaneously. To determine if the cytokines that induced fibroblast IL-l-a also induced fibroblast IL-l-p, we determined whether supernatants and lysates from unstimulated and cytokine-stimulated fibroblasts contained IL-l-p bioactivity or IL-l-p protein. Il-l-p bioactivity was not detected in supernatants or lysates from unstimulated cells or cells stimulated with rIL-1 and/or rTNF (data not shown). IL-l-P protein was unable to be detected in supernatants or lysates of unstimulated fibroblasts or fibroblasts stimulated with rIL-1 or rTNF (data not shown). Similarly, IL-l-p protein was not detected in supernatants from cells stimulated with rIL-1 and rTNF in combination (data not shown). However, the IL-l-p prohormone was easily detected in lysates of cells stimulated with rIL-1 and rTNF in combination (Fig 5). Thus, fibroblasts produce IL-l-p in response to inflammatory cytokines. However, even when stimulated with rIL-1 and rTNF in combination, they only produced the intracellular prohormone of this cytokine. buy ortho tri-cyclen
IL-6 Production
IL-6 is often produced in association with IL-1 and IL-1 and IL-6 can synergize in regulating target cell function. To determine whether fibroblasts produce IL-6 in response to rIL-1 and/or rTNF, we determined whether supernatants from unstimulated and cytokine-stimulated fibroblasts contained IL-6 bioactivity or IL-6 protein. Bioactivity was assessed by determining if the supernatants stimulated the 3H-Tdr incorporating IL-6 dependent B9 plasmacytoma cells. Supernatants from unstimulated fibroblasts did not contain IL-6 bioactivity or IL-6 protein. In contrast, rIL-1 (a or P) and rTNF individually stimulated IL-6 production (Table 3). Importantly, fibroblasts incubated with rIL-1 and rTNF in combination produced far more IL-6 than the sum of the IL-6 production of cells stimulated with these cytokines individually (Table 3). This synergistic interaction was dose-dependent for both cytokines, was negated by preincubating either cytokine with its respective neutralizing antibody, and could not be reproduced by combining rIL-1 or rTNF with rIFN-7.
Figure 5. Demonstration of the IL-1-|3 in lysates of unstimulated and cytokine stimulated fibroblasts. Fibroblasts were labelled with “S-methionine for 24 hours in the presence and absence of rIL-1 (2.5 ng/ml) and/or rTNF (20 ng/ml) as noted. The IL-l-p protein in their lysates was then precipitated with antiserum against rIL-l-p and analyzed by SDS-PAGE and autoradiography. The arrow highlights the pro-IL-l-p. (Reproduced with permission.)
Table 3—Recombinant Cytokine Regulation of Fibroblast Interleukin-6 Production
Fibroblast Culture Conditions |
B9 Cell 3 H-Tdr Incorporation |
Control |
3,179 ±313 |
rIL-1 |
19,471 ±309 |
rTNF |
7,374 ±4,040 |
rIL-1 + rTNF |
49,515 ±4,750 |
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