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Astronauts live and work in relatively crowded, confined environments on the Space Shuttle and the International Space Station. They experience a unique set of stressors that contribute to a diminishment of many immune responses. This study investigated the ability of the shuttle crew members' monocytes to respond to gram-negative endotoxin that they could encounter during infections. Blood specimens were collected from 20 crew members and 15 control subjects 10 days before launch, 3 to 4 h after landing, and 15 days after landing and from crew members during their annual medical examination at 6 to 12 months after landing. When challenged with gram-negative endotoxin, the crew member's monocytes collected at all three time points produced lower levels of interleukin-6 (IL-6) and IL-1beta and higher levels of IL-1ra and IL-8 compared to those of control subjects. Cytokines were assessed by measuring the number of cells positive for intracellular cytokines. These values returned to normal 6 to 12 months after landing, except for IL-1ra, which was still higher (five- to sixfold) than in controls. This phenomenon was accompanied by an increased expression of Toll-like receptor 4 and decreased expression of CD14 on the crew members' monocytes at all time points. There were also increased levels of the lipopolysaccharide binding protein in the plasma of the crew members 3 to 4 h and 15 days after landing. This study shows that spaceflight-associated factors (in-flight and preflight) modulate the response of monocytes to gram-negative endotoxins.

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.

It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percent inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (mean +/- standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C(4) column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.

Neutropenia is considered a significant risk factor for invasive aspergillosis but is almost always associated with concurrent thrombocytopenia. Studies determined that platelets, like neutrophils, attached to cell walls of the invasive hyphal form of Aspergillus fumigatus. Organisms were damaged as shown by loss of cell wall integrity in scanning laser confocal microscopy and release of defined hyphal surface glycoproteins. Rapid expression appearance of surface antigen CD63 and release of markers of platelet degranulation confirmed activation during attachment to hyphae. Optimal platelet activation required opsonization of hyphae with fresh or heat-inactivated whole plasma. These effects of opsonization with whole plasma could not be duplicated by pooled human serum, immunoglobulin G, or fibrinogen, whether used separately or combined. Thus, platelets in the presence of whole plasma have the potential to play an important role in normal host defenses against invasive aspergillosis.

CD14 is a signaling receptor for both gram-negative bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM) that lacks terminal mannosyl units (AraLAM). In contrast, terminally mannosylated LAM (ManLAM) binds the macrophage mannose receptor (MMRc), although the ability of the MMRc to serve as a signaling receptor has not been previously reported. We compared the abilities of AraLAM and ManLAM to induce distinct responses in two monocytic cell populations, freshly isolated human peripheral blood monocytes (PBM) and monocyte-derived macrophages (MDM). The responses examined were chemotaxis and transient changes in free cytosolic calcium ([Ca2+]in). We found that AraLAM but not ManLAM was chemotactic for both PBM and MDM. Migration of these cells in vitro to AraLAM was specifically blocked by an anti-CD14 monoclonal antibody, suggesting that CD14 mediates the chemotactic response to AraLAM. Subsequently, we found that AraLAM induced a transient rise in [Ca2+]in levels within a subpopulation of PBM but not MDM. This response was blocked by anti-CD14 antibodies. In contrast, ManLAM induced a transient rise in [Ca2+]in levels within a subpopulation of MDM but not PBM. This response was blocked by either anti-CD14 or anti-MMRc antibodies. These data suggest that the MMRc can serve as a signaling receptor and that coligation of both CD14 and the MMRc is required to elicit a specific response. Thus, one response to LAM (chemotaxis) can be elicited solely by engaging CD14, whereas a different response (changes in [Ca2+]in levels) depends on both the differentiation state of the cells and concomitant engagement of CD14 and the MMRc.

The cytochrome b 245 of human neutrophils was reduced when the neutrophils under anaerobic conditions were stimulated by serum-treated zymosan, immune complexes, or phorbol myristate acetate. Reintroduction of oxygen rapidly reoxidized the cytochrome; azide was without effect on any of these reactions. This indicates that cytochrome b 245 participates in the respiratory burst of neutrophils.