Objectives Endotoxin induced suppression of cellular immune function is thought to contribute to septic complications in trauma patients. A rabbit model of endotoxemia was used to determine the relative roles of the anti-inflammatory factors interleukin-4 (IL-4), interleukin-10 (IL-10), transforming growth factor beta1 (TGFbeta1), and prostaglandin E sub 2 (PGE2) in addition to other factors, in inducing immunosuppression. Design T-cell suppressive factors (TSF) in serum ultrafiltrates were separated and tested for the presence of the known suppressive factors PGE sub 2, IL-4, IL-10, and TGFbeta1. Material and Methods New Zealand rabbits were injected with 50 microg/kg of purified Escherichia coli lipopolysaccharide. Animals were exsanguinated after 48 hours and serum was separated by ultrafiltration (cutoff 50 kd), TSK HW-40 size exclusion chromatography, and Q-Sepharose anion exchange chromatography. TSF activities of chromatographic fractions and serum samples were measured with a miltogen induced in vitro T-cell proliferation assay. Levels of PGE2, IL-4, IL-10, and TGFbeta1 were measured with enzyme immunoassays. Measurements and Main Results Serum TSF activity, and levels of PGE sub 2, IL-4, IL-10, and TGFbeta1 were increased after endotoxemia. Size exclusion chromatography revealed three major fractions (TSF1 sub -3) with up to 600 times more TSF activity compared with controls. IL-4 and IL-10 were found in TSF1 and TSF3. Further separation of TSF1 by anion exchange chromatography revealed a total of eight different T-cell suppressive factors. TGFbeta1 probably remained in the retentate after ultrafiltration, while PGE2 eluted at a higher retention time. The known anti-inflammatory factors TGFbeta1, IL-10, IL-4, and PGE2 only accounted for 13% of the total serum TSF activity of 614 U/mL. Conclusions Lipopolysacchoride shock results in the release of multiple T-cell suppressive factors in addition to known immunosuppressive factors, all of which contribute to the antiinflammatory response.
Sheep are often used to study tissue damage following shock after traumatic injury and in the course of other diseases. The processes involved are thought to be caused at least in part by elastase released from polymorphonuclear leukocytes (PMNs). Since little is known about elastase and its role as a mediator of tissue damage in sheep, we studied the biochemical properties and release characteristics to sheep leukocyte elastase (SLE) in comparison of those of human leukocyte elastase (HLE). Both enzymes showed similar molecular masses, amino-acid compositions, N-terminal amino-acid sequences, and abilities to digest elastin substrates. Differences, however, were found in kinetic parameters measured with the elastase-specific substrate N-methoxysuccinyl-(L-alanyl)2-L-prolyl-L- valine-4-nitroanilide (MeoSuc-AAPV-pNa). The Michaelis constant (Km) of ovine elastase was nearly 10 times higher (1.82 mM) than the Km of HLE (0.21 mM). Values of SLE calculated for kcat were 70% and for kcat/Km 8% of corresponding values determined for HLE. In addition, significant differences between sheep and human PMNs were found in in vitro stimulation experiments. In contrast to human PMNs, sheep neutrophils released no active elastase, and only 50 to 70% of the H2O2 produced by human PMNs. This failure to release active elastase could not be explained by a lower elastase content of sheep PMNs, as there were no significant differences found between the elastase contents of sheep and human PMNs. We conclude that elastase liberated by stimulated sheep PMNs is inactivated by a concomitantly released proteinase inhibitor also located within the sheep PMNs.(ABSTRACT TRUNCATED AT 250 WORDS)
