Lennart Hänström

Caspase 1 Involvement in Human Monocyte Lysis Induced by Actinobacillus actinomycetemcomitans Leukotoxin

ABSTRACT Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of ≥5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Abundant Secretion of Bioactive Interleukin-1beta by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

ABSTRACT Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1β, while the effects on IL-6 and TNF-α production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1β than did LPS (100 ng/ml). The secreted IL-1β was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1β. The presence of specific antibodies to IL-1β or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1β. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1β by human macrophages, which is mediated by activation of caspase 1.

IL-1β and TNF-α Regulate IL-6-type Cytokines in Gingival Fibroblasts

Interleukin-6 (IL-6)-type cytokines are pleiotropic molecules capable of stimulating bone resorption and expressed by numerous cell types. In the present study, we tested the hypothesis that gingival fibroblasts may exert local osteotropic effects through production of IL-6 and related cytokines. IL-6-type cytokine expression and regulation by IL-1β and tumor necrosis factor-α (TNF-α) were studied in fibroblasts from the non-inflamed gingiva of healthy individuals. Constitutive mRNA expression of IL-6, IL-11, and leukemia inhibitory factor (LIF), but not of oncostatin M (OSM), was demonstrated, as was concentration-dependent stimulation of IL-6 and LIF mRNA and of protein by IL-1β and TNF-α. IL-11 mRNA and protein were concentration-dependently stimulated by IL-1β. The signaling pathway involved in IL-6 and LIF mRNA stimulation involved MAP kinases, but not NF-κB. The findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic IL-6-type cytokine production mediated by activation of MAP kinases. Abbreviations: IL-1α (interleukin-1α); IL-1β (interleukin-1β); IL-6 (interleukin-6); IL-11 (interleukin-11); LIF (leukemia inhibitory factor); OSM (oncostatin M); α(1)-coll. I (α(1)-collagen I); ALP (alkaline phosphatase); BMP-2 (bone morphogenetic protein-2); OC (osteocalcin); BSP (bone sialoprotein); TNFR I (tumor necrosis factor receptor I); TNFR II (tumor necrosis factor receptor II); IL-1R1 (interleukin-1 receptor 1); GAPDH (glyceraldehyde-3-phosphate dehydrogenase); RPL13A (ribosomal protein L13A); mRNA (messenger ribonucleic acid); cDNA (complementary deoxyribonucleic acid); PCR (polymerase chain-reaction); BCA (bicinchoninic acid); ELISA (enzyme-linked immunosorbent assay); α-MEM (α modification of Minimum Essential Medium); and FCS (fetal calf serum).

Interleukin-4 and interleukin-13 inhibit the expression of leukemia inhibitory factor and interleukin-11 in fibroblasts

Cytokines produced by inflammatory or resident mesenchymal cells play important modulatory roles in the pathogenesis of inflammation induced bone loss. In the present study, the effects of IL-4 and IL-13 on the expression of three osteotropic cytokines in the IL-6 family expressed in human gingival fibroblasts were studied. IL-4Rα and IL-13Rα1 mRNA were constitutively expressed in human gingival fibroblasts. The inflammatory cytokines IL-1β and TNF-α increased expression of IL-6, LIF, and IL-11 mRNA and protein in the gingival fibroblasts. Addition of IL-4 or IL-13 had no effect on IL-6 expression, but significantly inhibited LIF and IL-11 mRNA and protein stimulated by IL-1β and TNF-α. No involvement of NF-κB or STAT1 was observed in the inhibition. STAT6 was phosphorylated at Y641 by treatment with IL-4 and knockdown of STAT6 with siRNA decreased the inhibition of IL-11 and LIF expression by IL-4 in IL-1β and TNF-α stimulated cells. This study suggests that activation of STAT6 by IL-4 and IL-13, through type 2 IL-4 receptors, inhibits production of IL-11 and LIF stimulated by IL-1β and TNF-α in human gingival fibroblasts. A negative modulatory role of IL-4 and IL-13 in osteotropic cytokine production could be a mechanism playing an important inhibitory role in inflammation induced periodontitis.

Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes

The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes using leakage of51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of51Cr, a high uptake of trypan blue, and total cell necrosis, as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range of 10–500 μg/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide.

The Effect of zinc Tape upon Wound Healing1

The healing of excisional wounds in rats, which were treated with zinc tape, gauze sponge or porcine skin, was studied. Wound closure was completed earlier in zinc-tape-treated wounds than in wounds treated with a gauze sponge or porcine skin. Wound contraction was more pronounced in gauze- and porcine skin-treated wounds than in zinc-tape-treated wounds. More foreign body giant cells were seen 14 days after operation in gauze-treated contra zinc tape-treated wounds, whereas hydroxyproline concentration was higher in tape-treated wounds. Histochemically the acid phosphatase activity was pronounced in macrophages, foreign body giant cells and fibroblasts. The alkaline phosphatase activity was pronounced in granulocytes and fibroblasts. Quantitatively a reduction in alkaline phosphatase activity was seen in gauze-treated wounds from 7 to 14 days. A decrease was seen in albumin concentration in gauze- as well as zinc-tape-treated animals which was most pronounced after 7 days in the gauze-treated animals.

The effect of diphenylhydantoin upon the biosynthesis and degradation of collagen in cat palatal mucosa in organ culture

Abstract The effect of diphenylhydantoin (DPH) upon collagen metabolism in cat palatal mucosa was studied in organ culture using a grid technique. The incorporation of [3H]proline into [3H]hydroxyproline was taken as a measure for collagen synthesis. Using a 24 hr [3H]proline pulse the collagen synthesis in the control explants was found to be constant from the third day until the end of the culture period at day 15. DPH, in the concentrations 5 and 20 μg/ml, did not influence collagen or non-collagen protein synthesis. When collagen synthesis was studied using continuous labelling with [3H]proline for 6 days, DPH increased the incorporation of radioactive collagen into the tissues. To study the collagen degradation cats were injected with [3H]proline and six days later the palatal mucosa was excised and cultured in the absence and presence of DPH for 6 days. The release of radioactive hydroxyproline into the culture medium was taken as a measure for collagen degradation. DPH (20 μg/ml) resulted in about a 50 per cent decreased release of radioactive and non-radioactive hydroxyproline to the medium. To study the effect of DPH on the degradation of in vitro labelled collagen, mucosal explants were cultured in the presence of [3H]proline for 24 hr, and post-cultured for another 24 hr period. The release of labelled and unlabelled hydroxyproline from the explants was then followed for 4 days. DPH was found to inhibit the release of radioactive hydroxyproline (from collagen 1–2 days old) only slightly, but caused a strong inhibition of the degradation of non-radioactive collagen. It is concluded that DPH is an inhibitor of collagen degradation and that the inhibition applies mainly to the degradation of older collagen.

The effect of zinc on bacterial phagocytosis, killing and cytoprotection in human polymorphonuclear leucocytes.

An in vitro study examining the effects of zinc treatment on human PMN cell phagocytosis and killing of Staphylococcus aureus and Staphylococcus epidermidis and the cytoprotection of zinc against staphylococcal toxins. Phagocytosis was studied by transmission electron microscopy using different microbiological techniques, one of which was designed to follow the kinetics of bacterial killing. No effect was found on phagocytosis and bacterial killing. The cytotoxic effects of a crude toxin and an α‐toxin extracted from Staphylococcus aureus preparations were studied on human PMN cells using the standard 51Cr release assay. Both toxins induced a dose‐dependent leakage of 51Cr, indicating cell membrane damage. These results were confirmed by electron microscopy during the phagocytosis of S. aureus, where severe PMN cellular degeneration was observed. The addition of zinc to PMN cells strongly inhibited the release of 51Cr. In conclusion, our results show that zinc in higher than physiological concentrations does not inhibit PMN cell functions such as phagocytosis and intracellular killing of S. aureus and S. epidermidis. The addition of zinc may be beneficial in certain clinical situations, such as wound healing, zinc deficiency and infections involving toxin‐producing bacteria, e.g. S. aureus.

The Effects of Phenytoin on Serum and Organ Concentrations of Zinc and Copper in Cat

Summary: The effects of phenytoin (PHT) on zinc (Zn) and copper (Cu) concentrations in serum and different organs have been studied in cats. Five cats were treated with PHT 5 mg/kg body weight each day. Increased serum concentrations of Zn and Cu were found after 12 weeks of treatment. Increased Zn concentrations were found in the kidney and increased Cu concentrations in the liver and kidney. There were signs of fatty degeneration of the liver. It is postulated that the drug acts as a chelator of the minerals and enhances their absorption from the intestine. A Cu accumulation may occur during PHT treatment.

Stimulation of bone resorption and cell proliferation in vitro by human gingival fibroblasts from patients with periodontal disease

In the present communication we report that fibroblasts, isolated from human gingiva obtained from 13 different patients, secreted soluble product(s) which can promote bone resorption in vitro. Fibroblasts were isolated from explants of human gingiva, subcultured, grown to confluent monolayers, subsequently cultured in growth arrest media for 0-72 h and conditioned media harvested. Bone resorption was assessed in cultured mouse calvarial bone by quantifying the mobilization of minerals and the release of lysosomal enzymes. Human fibroblast-conditioned media (HFCM) dose-dependently stimulated the release of 45Ca from prelabelled bones and the mobilization of stable calcium and inorganic phosphate from unlabelled bones. In addition, HFCM increased the release of beta-glucuronidase and beta-N-acetylglucosaminidase from the calvaria. No effect of HFCM on the release of 45Ca from dead bones could be seen. HFCM caused a dose-dependent increased degradation of bone matrix proteins, as assessed by the release of 3H from [3H]proline-labelled calvaria. The stimulation of 45Ca release could already be seen after 3-12 h of treatment. Treatment of the bones with HFCM for 12 h was sufficient to obtain a prolonged stimulation of 45Ca release. Bones cultured in the presence of HFCM showed an increased number of osteoclasts. Calcitonin, but not indomethacin, inhibited 45Ca release stimulated by HFCM. Ultrafiltration of HFCM did not cause any loss of the 45Ca release response. The amount of bone-resorbing activity produced by the gingival cells was proportional to the number of cells. In addition, HFCM stimulated the proliferation of human fibroblasts and osteoblast-enriched mouse calvarial bone cells. It is concluded that human gingival fibroblasts secrete one or several factors that can stimulate osteoclastic bone resorption in vitro by a prostaglandin-independent pathway.