Per Follin

Intravenous Immunoglobulin G Therapy in Streptococcal Toxic Shock Syndrome: A European Randomized, Double-Blind, Placebo-Controlled Trial

The efficacy and safety of high-dose intravenous polyspecific immunoglobulin G (IVIG) as adjunctive therapy in streptococcal toxic shock syndrome (STSS) were evaluated in a multicenter, randomized, double-blind, placebo-controlled trial. The trial was prematurely terminated because of slow patient recruitment, and results were obtained from 21 enrolled patients (10 IVIG recipients and 11 placebo recipients). The primary end point was mortality at 28 days, and a 3.6-fold higher mortality rate was found in the placebo group. A significant decrease in the sepsis-related organ failure assessment score at days 2 (P=.02) and 3 (P=.04) was noted in the IVIG group. Furthermore, a significant increase in plasma neutralizing activity against superantigens expressed by autologous isolates was noted in the IVIG group after treatment (P=.03). Although statistical significance was not reached in the primary end point, the trial provides further support for IVIG as an efficacious adjunctive therapy in STSS.

The Transcriptional Activation Program of Human Neutrophils in Skin Lesions Supports Their Important Role in Wound Healing

To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin lesions. After migration to skin lesions, PMNs demonstrated a significant transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming of PMNs. Among the up-regulated genes were cytokines and chemokines critical for chemotaxis of macrophages, T cells, and PMNs, and for the modulation of their inflammatory responses. PMNs in skin lesions down-regulated receptors mediating chemotaxis and anti-microbial activity, but up-regulated other receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing. These included the breakdown of fibrin clots and degradation of extracellular matrix, the promotion of angiogenesis, the migration and proliferation of keratinocytes and fibroblasts, the adhesion of keratinocytes to the dermal layer, and finally, the induction of anti-microbial gene expression in keratinocytes. Notably, the up-regulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing.

Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told.

The paradigm for activation of the neutrophil NADPH oxidase with the protein kinase C activator phorbol myristate acetate (PMA) states that the oxidase assembles in the plasma membrane and that the metabolites generated are released extracellularly. This paradigm is challenged by the results presented. Most of the PMA‐induced oxidase activity measured as chemiluminescence and dichlorofluorescein fluorescence was insensitive to scavengers of superoxide anion and hydrogen peroxide. This indicates that oxidase activity also takes place in a cellular compartment that the scavengers cannot reach. From the results obtained with granule‐deficient HL‐60 cells and cord blood neutrophils, we suggest that the scavenger‐insensitive part of the NADPH oxidase activity in normal neutrophils re‐sides in an intracellular compartment identical to or originating from granules. Our results also indicate that specific and azurophil granules have to be in very close contact to allow the generated oxygen metabolites to reach and react with myeloperoxidase.

Mechanisms in Neutrophil Priming: Characterization of the Oxidative Response Induced by Formylmethionyl-Leucyl-Phenylalanine in Human Exudated Cells

Exudate human polymorphonuclear neutrophils were isolated and investigated regarding oxidative responsiveness and priming ability. The exudate neutrophils were found to produce an increased amount of O2‐ and H2O2 when stimulated with formylmethionyl‐leucyl‐phenylalanine (fMLP). i.e. these cells were metabolically primed. Cytochalasin B (cyl B) pretreatment affected the production of O2‐ by exudate cells, although to a lesser extent than the production by peripheral blood cells, in which a substantial increase was induced. Addition of N‐ethylmaleimide (NEM) to activated exudate and peripheral blood cells revealed no difference in oxidase in activation rate. To induce further priming, the cells were incubated in vitro with a synthetic diacylglycerol (sn‐1,2‐didecanoylglycerol: diC10), or the Ca2+ ionophore ionomycin. Results of this procedure showed significant differences between exudate and peripheral blood neutrophils: the peripheral cells expressed a primed response, which was measured as increased fMLP‐induced O2‐ production following incubation with both these substance: where a the metabolic activity of exudated cells was not affected by diC10 but was significantly primed by ionomycin (P<0.01). The exact route for diacylglycerol priming is unknown. However, our results with human neutrophils primed during exudation indicate an exhausted diC10‐priming pathway, with a retained sensitivity for priming [Ca2+]i rises.

Relationship between intracellularly and extracellularly generated oxygen metabolites from primed polymorphonuclear leukocytes differs from that obtained from nonprimed cells.

The ability of primed human polymorphonuclear leukocytes (PMNLs) to respond metabolically to stimulation with formylmethionyl-leucyl-phenylalanine (FMLP) was investigated. Cells isolated from an aseptic inflammatory reaction and from patients with a severe bacterial infection as well as cells that had been treated with a bacterial lipopolysaccharide were investigated. When these cells were compared to peripheral blood cells isolated from healthy controls, they were found to be metabolically primed, i.e., the cells gave rise to an increased chemiluminescence response to subsequent stimulation with the peptide. It was also shown that proportionally more of the activity generated from the primed PMNL was of an intracellular origin compared with that obtained from nonprimed cells. The biological effects induced by radicals produced extracellularly and intracellularly are discussed.

Human neutrophil chemiluminescence and f‐Meth‐Leu‐Phe receptor exposure in bacterial infections

Polymorphonuclear leukocytes were isolated from 12 patients with acute bacterial infections and the ability of the chemoattractant formylmethionyl‐leucyl‐phenylalanine (FMLP) to bind and induce a metabolic response in these cells was investigated. Cells isolated from the patients showed a significantly increased metabolic response in a luminol enhanced chemiluminescence assay compared to cells, isolated and analyzed in parallel, from healthy controls i.e. the patient cells were primed. The primed state was, as calculated by Scatchard analysis, accompanied by a significantly increased number of FMLP receptors exposed on the cell surface while the receptor binding affinity remained unchanged. There was, however, no correlation between the degreee of priming and the degree of receptor upregulation. Furthermore, it was found that stimulation also with phorbol myristate acetate (PMA), a substance lacking specific cell surface receptors on the PMNL, gave rise to an increased metabolic response in the primed cells. These results indicate that the priming activity induced by a bacterial infection can only partly be explained by receptor modulation and that other mechanisms must also be considered. This study was approved by the Ethics Committee of the Medical Faculty, University of Linköping.

Phagocytosis following Translocation of the Neutrophil b‐Cytochrome from the Specific Granule to the Plasma Membrane is Associated with an Increased Leakage of Reactive Oxygen Species

The effect of neutrophil b‐cytochrome translocation on the respiratory burst activation generated during phagocytosis of yeast particles was investigated. Secretion of neutrophil specific granules was induced by the calcium ionophore ionomycin prior to phagocytosis. The secretory process is associated with a translocation from the specific granules to the plasma membrane of the respiratory burst b‐cytochrome. Respiratory burst activity was measured as release of hydrogen peroxide in the absence of azide (extracellular leakage) and in the presence of azide (total produclion). The subcellular localization of the b‐cytochrome was found to affect the extracellular release of hydrogen peroxide in that a plasma membrane localization was associated with a significantly increased release during phagocytosis. It should be pointed out, however, that most of the hydrogen peroxide, both in control and in ionomycin‐treated cells, is produced intracellularly, probably in the phagosomes.

Phagocytosis by lipopolysaccharide-primed human neutrophils is associated with increased extracellular release of reactive oxygen metabolites

The effect of priming human neutrophils with lipopolysaccharide was investigated regarding the respiratory burst activity generated during phagocytosis of IgG-or C3b-opsonized yeast particles. LPS pretreatment significantly enhanced the respiratory burst activity, measured as luminol-amplified chemiluminescence, of both types of opsonized particles. In control cells most of the activity was produced intracellularly, probably in the phagosomes. In the primed cells, however, extracellular release of reactive oxygen metabolites was significantly increased during Fc-and CR3-mediated phagocytosis (P < 0.01 andP < 0.002, respectively). The release was most pronounced when using C3b-opsonized particles. Potent oxygen metabolites acting together with lysozomal enzymes are of importance in inflammatory-induced tissue damage. An increased extracellular release of reactive oxygen species by phagocytizing primed neutrophils can therefore lead to greater damage to the surrounding tissues.

Altered O2−H2O2 production ratio by in vitro and in vivo primed human neutrophils

Human neutrophils were primed by exudation or pretreatment with a synthetic diacylglycerol (diC10), the Ca2+ ionophore ionomycin or lipopolysaccharide (LPS). Compared to control cells, these primed cells showed a significantly decreased O2-/H2O2 ratio when stimulated with formylmethionyl-leucyl-phenylalanine (FMLP). This shift indicates a comparative (and net) increased H2O2 detection in the extracellular medium and can not be explained by a dose-dependent impairment in either O2- or H2O2 detecting capacity. An altered H2O2 degenerating capacity was not observed in the primed cells. We propose that priming enhances the capacity to divalently reduce oxygen and thereby directly produce H2O2.

Degranulation in human neutrophils primes the cells for subsequent responsiveness to the chemoattractant N-formylmethionylleucylphenylalanine but does not increase the sensitivity of the NADPH-oxidase to an intracellular calcium rise

Both the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) and the calcium-specific ionophore ionomycin can activate the NADPH-oxidase in human neutrophils. However, since ionomycin and FMLP activity differ in their requirement for azide, a potent inhibitor of the hydrogen peroxide consuming enzymes catalase and myeloperoxidase, we propose that the two stimuli can activate different pools of the oxidase. Degranulation, induced in vitro by sn-1,2-dedecaoylglycerol or in vivo by an exudation process, resulted in a priming of the cells using FMLP as stimulating agent as well as in a reduced capacity to generate H2O2 in response to ionomycin. The sensitivity of the plasma membrane-bound NADPH-oxidase to an intracellular [Ca2+] rise, induced by the ionophore was, however, not changed by the degranulation. From these results we propose that FMLP activates the plasma membrane-bound oxidase, whereas the ionophore is capable of activating a granule-bound pool of the oxidase.