Tetracycline inhibition of neutrophil-associated collagenolysis has been the focus of a number of investigations. Evidence has suggested that this inhibition results from the ability of this family of antimicrobial drugs to bind divalent cations such as Ca2+ and Zn2+, two cations that are required for full expression of activity of metalloproteinases such as collagenase and gelatinase. Data presented in this study demonstrate that tetracyclines can also inhibit neutrophil-mediated RBC lysis, superoxide anion synthesis, degranulation and migration. To some extent, tetracycline inhibition of neutrophil functions is mimicked by the Ca2+ binding agents, EDTA and TMB-8. However, Ca2+ enrichment restored full function to EDTA- and TMB-8-treated cells but not to tetracycline-treated neutrophils. This suggests that Ca2+ binding plays a role but is not the critical effect leading to tetracycline suppression of neutrophil functions. It has been suggested that tetracyclines can suppress leukocyte-associated tissue damage. Host tissues are protected from neutrophil-mediated damage by two mechanisms: 1. Neutrophil granule-associated enzymes are secreted in an inactive state; and, 2. tissues are protected from these enzymes by a potent inhibitor shield. Neutrophils can bypass these protective elements by activating enzymes and by destroying the shield through the synthesis of oxygen radicals. Therefore, tetracyclines may suppress neutrophil-mediated tissue damage by inhibiting their migration and degranulation and, potentially more importantly, by suppressing synthesis of oxygen radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Background and Purpose Activated leukocytes appear to potentiate central nervous system reperfusion injury, and agents that block leukocyte adhesion have shown neuroprotective efficacy in experimental models. Doxycycline, a tetracycline antibiotic, inhibits leukocyte function in vitro, presumably through divalent cation binding. We used a model of focal central nervous system reperfusion injury to determine the efficacy of doxycycline treatment in preserving neurological function.Methods Rabbits randomly received 10 mg/kg IV doxycycline 30 minutes before ischemia (pretreatment group) or 45 minutes after ischemia (posttreatment group) or received phosphate-buffered saline vehicle (control group) followed by 10 mg/kg q 8 hours times two. The average length of reversible spinal cord ischemia required to produce paraplegia (P M ) at 18 hours was calculated for each group.

Human periodontal ligament-derived fibroblasts (HPLF) were grown to confluency in culture and were subjected to various combinations of neutrophils (PMNs), lipopolysaccharide (LPS) and the chemoattractant formylmethionyl-leucyl-phenylalanine (FMLP). After treatment, the cells were stained to distinguish between normal and damaged cells. The stain also allowed an estimation of PMN adherence to the HPLF monolayer. We report that FMLP, LPS or PMNs alone did not damage HPLF cells, nor did PMNs when combined with LPS or FMLP separately. However, PMNs subjected to combinations of LPS (10-1000 ng/ml) and FMLP (10(-9)-10(-6) M) caused significant PMN-mediated fibroblast damage. LPS concentrations greater than 1000 ng/ml inhibited the cytotoxic reaction. Furthermore, we found that FMLP alone did not significantly enhance PMN adherence to the HPLF monolayer but that LPS increased PMN adherence 3-fold and the combination of LPS and FMLP enhanced adherence 6-fold. We conclude that LPS promotes PMN adherence to fibroblasts and that such adherence appears to be a crucial, but insufficient stimulus, for the induction of PMN-mediated HPLF injury.

Chlorhexidine was cytotoxic to both neutrophils and red blood cells over a narrow concentration range of 0.01 to 0.02% drug. Serum provided significant protection against the cytotoxic effects of the antimicrobial agent. At a concentration of 0.01%, chlorhexidine acted as a potent activator of the neutrophil's oxidative burst, stimulating the cells to produce oxygen radicals such as superoxide (O2). Chlorhexidine ranging from 10 ' to 10~-caused spontaneous degranulation of neutrophils. If neutrophils were pretreated with chlorhexidine and then activated by the chemoattractant tripeptide FMLP, chlorhexidine inhibited the induced O2 generation and degranulation. If, on the other hand, neutrophils were activated with phorbol myristate acetate (PMA) following chlorhexidine treatment, the antimicrobial agent enhanced both 02~ synthesis and degranulation. It appears that the responsiveness of chlorhexidine-treated neutrophils to subsequent activators is dependent upon the nature of the activator.

The in vitro migration of human polymorphonuclear neutrophils (PMNs) was studied employing an enzymatic assay of cell migration with phorbol myristate acetate (PMA) as the test stimulant. Our data clearly show that PMA in concentrations between 1 and 100 ng/ml in the lower wells of blind-well chambers induced chemotactic migration. Chemokinesis (increased migration) was not induced when PMA was present in both the upper and lower chambers (i.e., in a nongradient mode). Clearly our data indicate that PMA is chemotactic for human PMNs and, coupled with published studies of the effect of PMA on PMNs, suggest activation of an intracellular gradient of membrane-associated protein kinase C as a possible new mechanism for the induction of oriented migration of PMNs. Such a mechanism may be generalized to include membrane-soluble materials (e.g., inflammatory mediators, microbial products), which establish internal gradients of activated PKC rather than via the "classic" agonist-surface receptor mechanism, providing an alternative pathway for the induction of leukocyte chemotaxis.