Robert D. Nelson

Staphylococcal and Streptococcal Pyrogenic Toxins Involved in Toxic Shock Syndrome and Related Illnesses

Toxic-shock syndrome (TSS) is an acute onset, multiorgan illness which resembles severe scarlet fever. The illness is caused by Staphylococcus aureus strains that express TSS toxin-1 (TSST-1), enterotoxin B, or enterotoxin C. TSST-1 is associated with menstrual TSS and approximately one-half of nonmenstrual cases; the other two toxins cause nonmenstrual cases, 47% and 3%, respectively. The three toxins are expressed in culture media under similar environmental conditions. These conditions may explain the association of certain tampons with menstrual TSS. Biochemically, the toxins are all relatively low molecular weight and fairly heat and protease stable. Enterotoxins B and C, share nearly 50% sequence homology with streptococcal scarlet fever toxin A; they share no homology with TSST-1 despite sharing numerous biological properties. Numerous animal models for development of TSS have suggested mechanisms of toxin action, though the exact molecular action is not known. The toxins are all potent pyrogens, induce T lymphocyte proliferation, requiring interleukin 1 release from macrophages, suppress immunoglobulin production, enhance endotoxin shock, and enhance hypersensitivity skin reactions. The genetic control of the toxins has been studied and suggests the exotoxins are variable traits. Some additional properties of TSS S. aureus which facilitate disease causation have been clarified.

Detection and spatial distribution of the beta 2 integrin (Mac-1) and L-selectin (LECAM-1) adherence receptors on human neutrophils by high-resolution field emission SEM.

We have developed a method utilizing high-resolution field emission SEM and backscatter electron imaging of immunogold for detection of cell adhesion receptors on the surface of unfixed human neutrophils, using indirect immunogold localization of specific murine monoclonal antibodies (MAb) to the cell adhesion receptors L-selectin (LECAM-1) and the beta 2 integrin (Mac-1). We have observed that these two receptor populations occupy different membrane domains on the surface of unactivated human neutrophils. LECAM-1 was observed to occur in clusters on the tips of microvilli or membrane ruffles and was seldom detected on the membrane of the cell body. On the other hand, Mac-1 was found mainly on the membrane of the cell body in unactivated neutrophils, either singly or in small clusters, and was only rarely encountered on microvilli or ruffles. In contrast, the distribution of Mac-1 on activated, spreading neutrophils was markedly increased (up-regulated) and occurred in clusters on both the membrane of the cell body and also of surface projections, i.e., microvilli and ruffles. The unique distributions of LECAM-1 and Mac-1 on the surface of unactivated human neutrophils, as observed by high-resolution LVSEM, confirm the spatial relationships of these receptor types as predicted by models for the attachment of circulating neutrophils to vascular endothelium and their emigration to sites of inflammation.

Candida mannan: chemistry, suppression of cell-mediated immunity, and possible mechanisms of action.

The ability of Candida albicans to establish an infection involves multiple components of this fungal pathogen, but its ability to persist in host tissue may involve primarily the immunosuppressive property of a major cell wall glycoprotein, mannan. Mannan and oligosaccharide fragments of mannan are potent inhibitors of cell-mediated immunity and appear to reproduce the immune deficit of patients with the mucocutaneous form of candidiasis. However, neither the exact structures of these inhibitory species nor their mechanisms of action have yet been clearly defined. Different investigators have proposed that mannan or mannan catabolites act upon monocytes or suppressor T lymphocytes, but research from unrelated areas has provided still other possibilities for consideration. These include interference with cytokine activities, lymphocyte-monocyte interactions, and leukocyte homing. To stimulate further research of the immunosuppressive property of C. albicans mannan, we have reviewed (i) the relationship of mannan to other antigens and virulence factors of the fungus; (ii) the chemistry of mannan, together with methods for preparation of mannan and mannan fragments; and (iii) the historical evidence for immunosuppression by Candida mannan and the mechanisms currently proposed for this property; and (iv) we have speculated upon still other mechanisms by which mannan might influence host defense functions. It is possible that understanding the immunosuppressive effects of mannan will provide clues to novel therapies for candidiasis that will enhance the efficacy of both available and future anti-Candida agents.

Cytokine-stimulated chemotaxis of human neutrophils in a 3-D conjoined fibrin gel assay

The ability of neutrophils to migrate through three-dimensional (3-D) tissues in response to chemical stimuli is critical to their host defense function. However, studies characterizing stimulated migration in vitro have been largely limited to two-dimensional (2-D) surfaces. In this study, we have employed direct observation methods to quantify human neutrophil migration in 3-D fibrin gel using time-lapse video microscopy and automated cell tracking methods. A novel 3-D conjoined gel assay was developed to establish experimentally quantifiable and theoretically predictable diffusion gradients of chemotactic factors. This assay was used to measure objective migration parameters, namely the random motility and chemotaxis coefficients, in response to the cytokine, interleukin-8 (IL-8). The random motility coefficient, mu, showed a biphasic dependence on IL-8 concentration with a maximum of 1.1 x 10(-8) cm2/s at 5 x 10(-8) M IL-8; no significant motility was observed in the absence of IL-8. We further established the dependence of cell orientation bias, phi, on the concentration and gradient steepness (i.e., specific gradient, SG) of IL-8. Results indicate that phi increases with increasing SG, provided the concentration is maintained sufficiently low, which we conjecture to result from minimizing IL-8 receptor down-regulation. The chemotaxis coefficient, chi, was maximum at an intermediate SG for both IL-8 concentrations studied. We also examined the applicability of this assay to estimate mu and chi from indirect measurements of chemotaxis, namely the simpler measurement of cell redistribution after a prescribed incubation time, as opposed to direct cell tracking measurements. By virtue of measuring chi, this is the first quantitatively objective study of mammalian cell chemotaxis in a physiologically relevant 3-D gel and, in particular, of neutrophil chemotaxis on any substratum in response to the physiologically relevant chemotactic factor, IL-8.

ANTI-INFLAMMATORY ACTION OF DAPSONE : INHIBITION OF NEUTROPHIL ADHERENCE IS ASSOCIATED WITH INHIBITION OF CHEMOATTRACTANT-INDUCED SIGNAL TRANSDUCTION

Dapsone has clinical utility as an anti‐inflammatory agent but the mechanism of this action remains unknown. We have previously reported that dapsone inhibits β2 integrin (CD11b/CD18) ‐mediated adherence of human neutrophils in vitro and now describe studies designed to discover how dapsone‐mediated inhibition of this neutrophil function occurs. Results indicate that dapsone interferes with the activation or function of the G‐protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli. They also show that dapsone‐mediated suppression of this pathway inhibits the generation of second messengers essential to the activation of β2 integrin molecules, as well as respiratory and secretory functions of neutrophils exposed to chemoattractants. We propose that the inhibition of chemoattractant‐induced signal transduction by dapsone suppresses neutrophil recruitment and local production of toxic respiratory and secretory products in the affected skin of dermatitis herpetiformis and other neutrophilic dermatoses. J. Leukoc. Biol. 62: 827–836; 1997.

Nicotine-induced release of elastase and eicosanoids by human neutrophils

We examined the direct effects of nicotine on a variety of neutrophil functions at concentrations achievable in lung and oral tissues from cigarette smoking. The results show dose-dependent suppression of chemotaxis and phagocytosis, and enhancement of degranulation and eicosanoid generation, but not superoxide production. Cell viability was not affected by the concentrations of nicotine used in these experiments, as shown by trypan blue dye exclusion and MTT assays. These results implicate nicotine as the ingredient in cigarette smoke responsible for inflammatory damage to lungs and oral tissues observed in cigarette smokers.

Neutrophils dysfunction during the course of intra-abdominal infection.

Twenty-four patients with intra-peritoneal infections were studied sequentially to evaluate neutrophil chemotaxis, spontaneous migration, and chemiluminescence. In six patients, infection was due to spontaneous disease processes and 18 others, infection was a sequel of intra-abdominal operation. In the patients studied prior to drainage, operation chemotaxis, spontaneous migration and chemiluminescence were all significantly depressed. Operation resulted in a further depression of these functions, and recovery of neutrophil migratory responses was delayed for two weeks. During the period of functional depression, 16 patients developed recurrent infections (nine episodes of intra-abdominal abscess, and 12 episodes of extra-abdominal infection). These infection were associated with a fall-off in neutrophil migratory and chemiluminescence responses prior to clinical evidence of infection. This study suggests that the delayed recovery of neutrophil function may be related to the recurrent infection seen in this patient population. Further, monitoring of neutrophil function in patients recovering from intra-abdominal infection may provide early evidence of recurrent infection.

Spatial distribution of L-selectin (CD62L) on human lymphocytes and transfected murine L1-2 cells

SummaryWe have examined the topographical distribution of L-selectin on surface membrane domains of human lymphocytes and murine L1-2 cells transfected to express human L-selectin. L-selectin was immunolocalized using murine monoclonal DREG 200 Fab antibody and a 12 nm colloidal gold-conjugated secondary antibody. Cell surface morphology and surface distribution of gold-labelled L-selectin were visualized using backscatter electron images obtained by high-resolution, field emission scanning electron microscopy. The topographical morphologies of lymphocytes of both types were complex. The surface of human lymphocytes was composed of both microvilli and ruffles; that of the murine cells was composed of long microvilli and few, if any, ruffles. L-selectin on human lymphocytes was observed primarily as focal clusters on the apical surfaces of ruffles and microvilli. Similarly, on the transfected murine cells, L-selectin was detected predominantly on the apical surface of microvilli. We conclude that L-selectin has a common spatial distribution and clustered organization on all leukocytes examined to-date, and that these features of receptor expression likely facilitate rolling of circulating leukocytes on the endothelial surface.

Mechanisms of the adjuvant action of hemoglobin in experimental peritonitis: 2. Influence of hemoglobin on human leukocyte chemotaxis in Vitro

Abstract The influence of hemoglobin on the chemotactic response of human polymorphonuclear neutrophils (PMNs) and monocytes under agarose was evaluated. Hemoglobin present at the site of the chemotactic factor, E. coli culture filtrate (BFE) or zymosan-activated serum (ZAS), in a concentration of 4% causes depression of the chemotactic response of cells of both types. The degree of depression of the response is directly proportional to the concentration of hemoglobin (2–10%) at the site of chemotactic factor. If hemoglobin is added to the cells, much lower concentrations (0.001–0.1%) are sufficient to elicit the same effect. Hemoglobin did not interfere with the spontaneous migration of the cells in this experiment unless its concentration at the site of the cells was higher than 0.1%. The presented evidence supports the idea that hemoglobin inhibits the chemotactic response of PMNs by interfering with the effective interaction of the cytotaxin to the receptor on the cell wall. These findings are consistent with the findings that the influx of PMNs into the peritoneal cavity in response to intraperitoneal bacterial inoculum is inhibited by hemoglobin and that the mechanism by which PMNs are prevented from responding to bacteria in vivo involves the interference by hemoglobin with the natural chemotactic response of PMNs.

Effects of detergents on proliferation and metabolism of human keratinocytes

Abstract Effects of low concentrations of detergents on cultured human foreskin keratinocytes were assessed in vitro. The viability and activity of keratinocytes were assessed by measuring reduction of a tetrazolium dye as an indicator of mitochondrial metabolism. The keratinocyte proliferative responses after incubation with detergents were assessed by a spectrophotometric assay employing crystal violet dye and a fluormetric assay determining total DNA content. Both the cationic detergent cetyltrimelhylammonium bromide [CTAB] and the anionic detergent sodium lauryl sulfate [SLS] showed toxic effects on keratinocytes at concentrations as low as 3 μg/mg, but SLS was less toxic. However, both SLS and CTAB activated keratinocytes at very low concentrations. Proliferalive activity and mitochondrial metabolism increased. Serum partially protected keratinocytes against toxic and stimulatory activities of both detergents. We suggest that detergents may directly damage keratinocytes and thereby produce irritant contact dermatitis, but activation of keratinocytes by low concentrations may also produce dermatitis, perhaps by causing keralinocvtes to release cytokines