Protective effects of doxycycline in mesenteric ischemia and reperfusion.

In rats splanchnic artery occlusion (SAO) of the celiac and superior mesenteric arteries for 1 hr., followed by a period of reperfusion initiates a severe form of circulatory shock, which typically leads to death within 2 hrs, characterized by multiorgan failure in which neutrophils play a central role. Doxycycline (Dc) pretreatment (10mg/kg; 2 hrs. prior to occlusion) significantly increased the survival time and leukocytes retention in circulation and decreased the hemoglobin levels in the contents of the small intestine. Dc did not influence serum alanine aminotransferase or amylase levels (marker enzymes for liver and pancreas damages respectively) at the time of death. Creatinine phosphate kinase, a marker of cardiotoxicity, was significantly increased in serum from Dc-treated rats. We conclude that pretreatment with Dc has a protective effect in the SAO rat model and this protection cannot be ascribed directly to a specific sparing influence on the liver, pancreas, or heart.

Reduction of central nervous system reperfusion injury in rabbits using doxycycline treatment.

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 i.v. 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 (P50) at 18 hours was calculated for each group. RESULTS: For the control group (n = 13), the P50 was 22.8 +/- 2.2 minutes; for the pretreatment group (n = 14), 35.5 +/- 2.4 minutes (P < .01; t = 3.8); and for the posttreatment group (n = 13), 31.4 +/- 4.2 minutes (not significant; t = 1.6). Doxycycline also attenuated postischemic decreases in vivo leukocyte counts and inhibited in vitro leukocyte adhesion. Therapeutic doxycycline levels at 24 hours were confirmed in the plasma and spinal cord. CONCLUSIONS: This significant protective effect suggests that doxycycline, a safe and readily available agent, may play a role in reducing clinical central nervous system reperfusion injury.

Doxycycline suppression of ischemia-reperfusion-induced hepatic injury.

Leukocytes, in particular polymorphonuclear neutrophils (PMNs), are believed to play a central role in ischemia-reperfusion (I/R)-induced tissue injury. Changes in endothelial cells occurring during ischemia promote PMN binding to these cells during reperfusion, which primers PMN synthesis of oxygen radicals and release of cytotoxic proteins. These events lead to vascular damage and subsequent tissue injury. Recently we have shown that doxycycline (Dc), a member of the tetracycline family of antibiotics, inhibits PMN superoxide (O2) synthesis and degranulation in vitro. It also suppresses PMN-mediated RBC, fibroblast, and endothelial cytotoxicity, properties of the drug that may make it of use to protect tissues from I/R-induced injury. In this study we demonstrate that Dc administration either prior to clamping of the portal circulation, or 1 h after the reperfusion, significantly suppressed liver damage as assessed by serum levels of a marker of hepatic injury, alanine aminotransferase (s-ALT). The reduction in s-ALT was not a result of reduced reflow in the Dc-treated rats as indicated by Evans' blue perfusion data. The findings suggest that Dc and possibly other tetracyclines may be of value in protecting tissues and organs from I/R-mediated damage even if the drug is given after the ischemic event has occurred.

Doxycycline reduction of F-actin content of human neutrophils and fibroblasts.

We and others have shown that tetracyclines inhibit leukocyte adherence, migration, and phagocytosis, functions presumed to involve actin microfilament metabolism. In this study we investigated the influence of a tetracycline (doxycycline, Dc) on neutrophil and fibroblast actin metabolism. Human neutrophils and fibroblasts were pretreated with Dc or cytochalasin B (cB), stimulated with either the chemotactic peptide FMLP or phorbol myristate acetate (PMA), and the changes in phalloidin conjugate, associated F-actin were followed microscopically or quantified fluorometrically. Doxycycline suppressed, in a dose-related manner, the rise in F-actin content of neutrophils that normally follows their activation with either FMLP or PMA. Cytochalasin B had a similar affect on actin microfilament synthesis. Incubation of fibroblasts in Dc led to a loss of actin microfilaments and caused flattened adherent cells to round up and detach. Both Dc and cB also inhibited Con A-induced acceptor capping on neutrophils, a phenomenon known to be dependent on the presence of intact actin microfilaments. The data show that both Dc and cB influence actin metabolism and suggest they do so by differing mechanisms.

The influence of doxycycline of the attachment of fibroblasts to gelatin-coated surfaces and its cytotoxicity.

Because of their antimicrobial and anti-inflammatory properties, tetracyclines have been used systemically and locally in the treatment of periodontal disease. This study was done to evaluate the influence of doxycycline (Dc), a tetracycline, on fibroblast adherence to a protein coated surface and its cytotoxicity. Periodontal ligament derived fibroblasts (PLDFs) were either: 1) preincubated with Dc (0 to 100 micrograms/ml) and then allowed to adhere to a gelatin-coated surface or 2) adherence was first established and then Dc added to the system. After an appropriate time the number of PLDFs adherent, released, or lysed was estimated by a lactic dehydrogenase (LDH) assay. Preincubation of PLDFs in 25 micrograms Dc/ml or higher concentrations significantly (P < 0.01) reduced the number of adherent cells. Fifty micrograms Dc/ml and higher doses significantly (P < 0.01) increased PLDFs cytotoxicity as measured by LDH release. The same trend was noted if PLDFs were allowed to adhere and then subjected to the drug. Microscopic observation of fluorescein diacetate/propidium iodide-stained cells showed that attached-spread cells pulled in, rounded up, and detached in proportion to the increased dose of Dc and the percentage of red-stained cytotoxic cells rose in a similar manner. The data suggested that Dc can be cytotoxic and may inhibit PLDFs adherence and spread along a substratum.

Phorbol myristate acetate induction of chemotactic migration of human polymorphonuclear neutrophils.

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.

Comparison of doxycycline and a chemically modified tetracycline inhibition of leukocyte functions.

Tetracyclines (Tc's) have the ability to inhibit neutrophil (PMN) functions which may be of value in the treatment of neutrophil-driven pathologic processes. However, long term use of Tc's frequently lead to emergence of antibiotic resistant strains of bacteria. A chemically modified analogue of Tc, 4DTc, having minimal antibiotic activity was compared to Dc, a member of the Tc family, for its ability to inhibit neutrophil functions. 4DTc was significantly less effective at inhibiting PMN superoxide synthesis, PMA-induced degranulation and PMN-mediated RBC lysis than was Dc. 4DTc and Dc were equally as effective inhibiting monocyte, but not PMN, adherence to gelatin-coated surfaces. When incubated in media containing varying 4DTc or Dc concentrations, Dc accumulated in RBC's and PMN's at levels 3 times greater than that found for similar media levels of 4DTc. The data suggest that the lesser ability of 4DTc to inhibit several PMN functions as compared to Dc may be related to its reduced intracellular accumulation. It also suggest that Tc inhibition of monocyte adherence may be more influenced by extracellular than intracellular processes.

The influence of divalent cations and doxycycline on iodoacetamide-inhibitable leukocyte adherence.

Leukocyte adherence, an event critical to host defense, is reported to be dependent upon divalent cations. To test whether leukocyte binding is more influenced by the availability of Ca2+ or Mg2+, neutrophils and mononuclear leukocyte adherence to surfaces coated with differing proteins, in medium containing varying concentrations of Ca2+ and/or Mg2+ and induced with either FMLP or PMA were assessed. To account for nonspecific leukocyte-substrata interactions the OD's of iodoacetamide-inhibited adherent cell systems were subtracted from the OD values of adherent cells of noninhibited systems. Values presented were derived from OD's of iodoacetamide-inhibitable leukocyte binding. PMA was a much more potent inducer of leukocyte adherence than was FMLP, stimulating all available neutrophils and monocytes to adhere. In contrast, FMLP induced adherence by roughly 1/4 of the available neutrophils and few, if any, monocytes. Roughly the same binding pattern was noted whether surfaces were coated with albumin, fibrinogen, fibronectin, gelatin or serum. EDTA but not EGTA significantly suppressed leukocyte binding suggesting Mg2+ was more involved in binding than was Ca2+. Little leukocyte adherence to a substrata occurred in the absence of divalent cations, while enrichment of the medium with Mg2+ was more influential on cell binding than was enrichment with Ca2+. Interestingly, doxycycline, a member of the tetracycline family of drugs which has been reported to inhibit Mg(2+)-dependent neutrophil functions, had a slight inhibitory effect on neutrophil adherence at low drug concentrations, while it enhanced binding at higher doxycycline concentrations. In contrast, the tetracycline inhibited monocyte adherence in a dose-related manner.

Concurrent lipopolysaccharide enhances chemotactic response of human polymorphonuclear leukocytes to bacterial chemotaxin.

Polymorphonuclear neutrophil (PMN) function is thought to be critical in resistance to infectious agents and this implies that the PMN must be able to migrate into, and to function in, environments that may have high levels of bacterial lipopolysaccharide (LPS). Therefore, we have evaluated the effect of LPS on the in vitro migration of PMNs. Our data reveal that the human PMN is resistant to the deleterious effects of high levels of LPS, that in high concentrations LPS is, itself, a direct chemoattractant for PMNs, and that PMN migration toward a bacterial chemotaxin is enhanced if LPS is also present. Such capabilities suggest that the PMN may be uniquely qualified to migrate into microenvironments that are rich in LPS.

Fluxes and accumulation of tetracyclines by human blood cells.

Tetracyclines (Tc's) have anti-inflammatory properties unrelated to their antibiotic activities. Their anti-inflammatory property, in part, results from the ability of members of this family of antibiotic to inhibit neutrophil functions. There are marked differences in the ability of different Tc's to suppress neutrophils which may relate to their ability to cross the plasma membrane. To gain insight into the mechanism of Tc inhibition of neutrophils and the reason for the differences in antineutrophil effect of Tc's, we studied the flux and sequestration of Tc's in blood cells. Using centrifugation and a dibutylphthalate scrubber system we found that doxycycline (Dc) was rapidly taken up by blood cells reaching intracellular concentrations several times that found in the medium. Dc also rapidly effluxed when antibiotic loaded cells were placed in drug free medium. While Ca2+, Mg2+ nor protein separately were effective inhibitors of Dc influx, when divalent cations and proteins were combined Dc uptake was markedly suppressed. Tc uptake by blood cells ranked Dc greater than chlortetracycline = tetracycline greater than oxytetracycline, a ranking similar to that reported for neutrophil inhibition by members of the Tc family, suggesting that intracellular accumulation of drug is an important facet of Tc suppression of neutrophil function.

Suppression of human neutrophil functions by tetracyclines.

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)

Potential roles of Mg2+ and Ca2+ in NADPH oxidase dependent superoxide anion synthesis by human neutrophils.

Recent studies have shown that, in cell free systems, Mg2+, but not Ca2+, is absolutely required for the generation of reducing equivalents by neutrophil NADPH oxidase. Apparently Mg2+ is required for binding of cytosolic protein(s) to the plasma membrane in forming a functional NADPH oxidase complex. Using intact neutrophils made permeable to Mg2+ and Ca2+ by A23187 it was found that Mg2+ was required for the generation of reducing equivalents by PMA stimulated cells while Ca2+ inhibited cytochrome c reduction. On the other hand, FMLP-induced neutrophils were unable to generate reducing equivalents unless both Ca2+ and Mg2+ were present in the media. Sequential addition of these cations indicated that Ca2+ was required for transduction of the FMLP-receptor generated signal to a point in the pathway where Mg2(+)-dependent synthesis of reducing equivalents occurred. This step, presumably, is the Mg2(+)-dependent completion of the NADPH oxidase complex. Finally, approximately 50% of the neutrophil's 2.19 fmoles (6.32 mM) of Mg2+ was found to be in a readily exchangeable pool and that pretreatment of neutrophils with either FMLP or PMA increased this pool by only 4-5%. This suggests there is an adequate pool of Mg2+ available to support completion of NADPH oxidase complex and that a stimulus-induced rise in free Mg2+ is not required to assist in the formation of the complex.

An enzyme-assessed microplate-assay for neutrophil adherence. I. IgA-induced adherence of human PMNs.

The binding of PMNs to extracellular matrix and cells is crucial to PMN host defense. Adherence mechanisms and the many families of molecules involved are major areas of study. We present here details of an enzyme-assessed microtiter plate assay for neutrophil adherence. This assay uses low numbers of cells (50,000/well) and permits analysis of several hundred wells in a short period of time, by using an ELISA reader. With this assay we observed 5- to 10-fold increases in the number of adherent human PMNs in response to nanogram amounts of LPS or as little as 5.0 micrograms/ml of aggregated IgA. Although fluoride blocked the LPS-induced adherence response, IgA-induced cell binding was largely unaffected.

Effect of fluoride on movement of concanavalin A-acceptor molecules of human neutrophils.

The effects of fluoride (F) on neutrophil protuberance formation and induced Con A acceptor molecule migration were assessed microscopically. Below 5 mM, F had little effect on acceptor migration, while it markedly inhibited formation of colchicine-induced protuberances. The anion also increased the rate at which preformed protuberances regressed. Since protuberance formation is enhanced by disassembly of microtubules, these data suggest that F promotes and/or stabilizes microtubule assembly. Microtubule assembly is favored by binding of GTP to tubulin subunits, while GDP binding favors disassembly of microtubules. Since F binds with GDP, forming a new complex that mimics GTP, the anion would be expected to enhance microtubule assembly. Over the same F concentration range, the anion failed to inhibit acceptor polarization, but did inhibit cytochalasin B-enhanced dispersion of prepolarized Con A acceptors, implying that, at low concentrations, F also affected microfilament cycling. Concentrations of F in excess of 5 mM inhibited acceptor migration as well as protuberance formation. At 20 mM, the anion abolished both events, yet at this same concentration F induced neutrophil superoxide generation and degranulation, suggesting that acceptor migration is not a prerequisite for these two neutrophil effector activities.

Fluoride activation of neutrophils: similarities to formylmethionyl-leucyl-phenylalanine.

Fluoride induced degranulation of both primary and specific granules from neutrophils pretreated with cytochalasin B. There was a similarity in the dependency on extracellular Ca2+ for fluoride- and for FMLP-stimulated O2- generation and degranulation. Pertussis toxin, but not cholera toxin, inhibited FMLP and fluoride activation of neutrophils, while neither toxin affected PMA activation of these cells. These results suggest that fluoride and FMLP activate neutrophils through a common Ca2+-dependent and pertussis toxin-sensitive pathway.

Aggregated human colostral sIgA stimulates delayed, non-complement-dependent, NBT reduction by human neutrophils.

Antibodies often alert polymorphonuclear neutrophils (PMNs) to the presence of pathogens. In a study to learn if secretory immunoglobulins can carry out this function, we observed that as little as 4 micrograms/ml of secreted human immunoglobulin A from colostrum (sIgA), in the absence of antigen, stimulated human PMNs to reduce nitroblue tetrazolium (NBT). NBT reduction was inhibited 71% by superoxide dismutase. Active complement pathways were not required since comparable activity was obtained in the presence of heat-inactivated serum. Aggregated forms of sIgA were much more stimulatory than nonaggregated dimeric sIgA. Such interaction between PMNs and sIgA could act in situ to enhance protection against infections of exposed body sites or could initiate inflammatory tissue damage.