Fluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migration; however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. J774 macrophages were labeled with either calcein-acetoxymethyl ester (calcein-AM) or 2′,7′-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, acetomethyl ester (BCECF-AM), then assayed for their ability to migrate to zymosan-activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, comparison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF-AM decreased the number of cells responding to ZAS, while calcein-AM had essentially no effect. Neither probe significantly affected the number of cells migrating to medium alone. The inhibitory effects of BCECF-AM on cell migration increased with probe concentration (0.1-1.0 microM) and cell fluorescence. Cell viability was unaffected by either probe. In contrast to the results obtained by visual counting, measuring fluorescence of migrated cells did not reveal a significant difference between the chemotactic response of macrophages labeled with BCECF-AM and those labeled with calcein-AM. These experiments indicated that fluorescent probes can affect the chemotactic response and that inhibitory activity of these probes may not be detected when chemotaxis is quantified solely by automated methods.
pdf at: http://onlinelibrary.wiley.com/doi/10.1002/cyto.990190412/pdf
Chicken soup has long been regarded as a remedy for symptomatic upper respiratory tract infections. As it is likely that the clinical similarity of the diverse infectious processes that can result in “colds” is due to a shared inflammatory response, an effect of chicken soup in mitigating inflammation could account for its attested benefits. To evaluate this, a traditional chicken soup was tested for its ability to inhibit neutrophil migration using the standard Boyden blindwell chemotaxis chamber assay with zymosan-activated serum and fMet-Leu-Phe as chemoattractants. Chicken soup significantly inhibited neutrophil migration and did so in a concentration-dependent manner. The activity was present in a nonparticulate component of the chicken soup. All of the vegetables present in the soup and the chicken individually had inhibitory activity, although only the chicken lacked cytotoxic activity. Interestingly, the complete soup also lacked cytotoxic activity. Commercial soups varied greatly in their inhibitory activity. The present study, therefore, suggests that chicken soup may contain a number of substances with beneficial medicinal activity. A mild anti-inflammatory effect could be one mechanism by which the soup could result in the mitigation of symptomatic upper respiratory tract infections.
Glycoprotein-340 (gp-340) was first identified as a surfactant protein (SP)-D–binding molecule purified from lung lavage of patients with alveolar proteinosis (Holmskov, et al., J. Biol. Chem. 1997;272:13743). In purifying SP-A from proteinosis lavage, we isolated a protein that copurifies with SP-A and SP-D and that was later found by protein sequencing to be gp-340. We have shown that soluble gp-340 binds SP-A in a calcium-dependent manner independent of the lectin activity of SP-A. To examine the functional significance of this interaction, we tested the ability of soluble gp-340 to block SP-A binding to and stimulation of the chemotaxis of alveolar macrophages. We found that gp-340 does not affect the binding of SP-A to alveolar macrophages over a wide range of SP-A concentrations, nor does it inhibit the ability of SP-A to stimulate macrophage chemotaxis. We also found that gp-340 alone stimulates the random migration (chemokinesis) of alveolar macrophages in a manner independent of SP-A–stimulated chemotaxis. These results suggest that gp-340 is not a cell-surface receptor necessary for SP-A stimulation of chemotaxis, and show that gp-340 can directly affect macrophage function.
The major goal of this study was to investigate the mechanisms that link the host response to a local infection in the peritoneal cavity with the development of sepsis and lung injury. Rabbits were infected by intraperitoneal inoculation of fibrin clots containing Escherichia coli at 108, 109, or 1010 cfu/clot. Physiologic, bacteriologic, and inflammatory responses were monitored, and the lungs were examined postmortem. At a dose of 108 cfu/clot the animals had resolving infection, and a dose of 109 cfu/clot resulted in persistent infection at 24 h, with minimal systemic manifestations. In contrast, inoculation of 1010 cfu/clot resulted in rapidly lethal local infection, with septic shock and lung injury. The onset of septic shock was associated with a paradoxical lack of identifiable polymorphonuclear leukocytes (PMN; neutrophils) in the peritoneal cavity. The absence of PMN in the peritoneum was due in part to lysis of intraperitoneal PMN, because the peritoneal fluids contained free myeloperoxidase and induced rapid death of normal rabbit PMN in vitro. Although most animals became bacteremic, only those with a severe systemic inflammation response developed lung injury. These data show that control of an infection in the first compartment in which bacteria enter the host is a critical determinant of the systemic response. Above a threshold dose of bacteria, failure of the local neutrophil response is a key mechanism associated with deleterious systemic responses. Bacteremia alone is not sufficient to cause lung injury. Lung injury occurs only in the setting of a severe systemic inflammatory response and an inadequate leukocyte response at the primary site of infection.
Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation.