Dale R. Tabor

Enhanced Production of Monokines by Canine Alveolar Macrophages in Response to Endotoxin-Induced Shock

Abstract The enhanced production of soluble mediators by alveolar macrophages may be responsible for promoting lung injury in canines administered endotoxin. One of the most prominent monokines, interleukin 1 (IL-1), has the potential to significantly influence the responses of host tissues. In this study we analyzed alveolar macrophages from canines that were experimentally administered endotoxin (AMEC) for their ability to produce IL-1. When concentrated AMEC supernatants from in vitro cultures were incubated with fresh C3H/HEJ thymocytes, a threefold greater incorporation of [3H]thymidine resulted as compared to the response produced by controls. Heat treatment of the experimental preparations ablated this difference. Conversely, the activity of AMEC intracellular lysates did not significantly differ from the controls. Silver-staining the preparations separated by SDS-PAGE revealed a low-molecular-weight species (17 kD) in the AMEC supernatant lane while a similar molecular distribution was absent in all of the control preparations examined. Moreover, using the L929 cell line in a cytolytic bioassay we found that these same AMEC supernatants also contained significantly elevated levels of tumor necrosis factor. Collectively, this study suggests that during endotoxin-induced canine lung injury, the alveolar macrophages generate soluble species that can substantially regulate the hosts cellular response. This activity in the canine lung may play a critical role in the development and/or maintenance of the pathology associated with exposure to endotoxin.

Intravenous immunoglobulin modulates human mononuclear phagocyte tumor necrosis factor-α production in vitro

ABSTRACT: Mononuclear phagocytes (MO) secrete tumor necrosis factor-α (TNF) in response to inflammatory stimuli, most notably the bacterial product lipopolysaccha-ride (LPS). Cross-linking of MO Fc receptors also induces TNF release. Immunoglobulin for i.v. use is currently being investigated for the treatment and prophylaxis of neonatal sepsis and for the treatment of various syndromes of autoimmune dysfunction in children and adults. We examined the in vitro effect of immunoglobulin-γ (IgG) on neonatal (cord blood) monocyte and adult MO TNF production. Kinetic studies were performed on MO incubated with IgG alone and on MO preincubated with IgG and stimulated with interferon-γ/LPS. Incubation of MO in IgG (1–25 g/L) for 2, 6, and 24 h did not stimulate TNF secretion or production. However, enhanced TNF secretion was detected in MO preincubated in IgG and subsequently stimulated with interferon-γ/LPS. TNF secretion by cord blood monocytes was increasingly enhanced by preincubation for 6 h with 1, 10, and 25 g/L IgG (2413.1 ± 1389.4, p < 0.05; 4070.4 ± 3069.2, p < 0.005; and 6383.7 ± 2982.2, p < 0.005 versus 1215 ± 575.9 ng/L, respectively, in cells preincubated in medium alone). Significant enhancement was also detected in cord blood monocytes preincubated in IgG for 2 h. TNF secretion by adult MO was similarly enhanced (6082.0 ± 1732.8, p < 0.05; 7158.8 ± 3938.2, p < 0.05; and 7302.7 ± 3451.4, p < 0.05 versus 3353.2 ± 2946.7 ng/L for 1, 10, and 25 g/L IgG, respectively, versus preincubation in medium alone). In additional experiments performed with Fc, Fab, and F(ab‘)2 fragments, only F(ab’)2 fragments yielded positive results. Northern analyses revealed increased levels of mRNA for TNF only when 25 g/L IgG were used for preincubation. Preincubation in the lower concentrations of IgG did not result in increased accumulations of TNF mRNA. Thus, IgG acts primarily posttranscriptionally to enhance interferon-γ/ LPS-induced TNF release in vitro.

Differential induction of macrophage GSIB4-binding activity.

Activated macrophages display a terminal galactopyranosyl group on their membrane surface that binds the lectin Griffonia simplicifolia–IB4 (GSIB4). Using FITC‐conjugated GSIB4, we examined the induction and subsequent expression of this corresponding marker on peritoneal macrophages from normal (NMO) and LPS‐treated (LPS MO) mice. Although the percentage of fresh LPS MO explants that bound GSIB4 was always higher when compared to the NMO counterparts, marker expression on the latter was readily enhanced by cufturing the cells in vitro either alone or with stimuli. Moreover, we found that an increase in this activity was promoted by either nonspecific phagocytosis of latex beads, or γ‐interferon (γ‐IFN) treatment. Further investigation showed that a prerequisite sequence of signal delivery to the macrophages was associated with maximal expression of the GSIB4 binding. When γ‐IFN treatment preceded latex bead ingestion, maximum GSIB4 binding occurred. Data obtained from using short‐term (1 hr) and long‐term (24 hr) exposure to latex beads showed that metabolic processing of induction signals was required to enhance the response over time. This yielded better GSIB4‐binding activity when responses to these pulses were analyzed in freshly explanted macrophages.

Macrophage tumoricidal mechanisms are selectively altered by prenatal chlordane exposure

Macrophages (mø) derived from mice treatedin utero with chlordane show a significant delay of tumoricidal induction activity. In this study, mø from chlordane-treated animals required a 48 hin vitro period of induction with interferon-ψ and lipopolysaccharide (IFN/LPS) before they could kill P815 targets. Similarly, mø from chlordane-treated animals also failed to produce an immediate H2O2 burst upon perturbation. Conversely, their stimulated control mø counterparts were tumoricidal by 2 h and exhibited a respiratory burst without any delay. Moreover, levels of the second messenger, inositol triphosphate (IP3), were significantly delayed in chlordane-treated animals following interaction with IFN/LPS. When nitrate/nitrite production was analyzed as an alternate mechanism for killing tumors, stimulated mø from both normal and chlordane-treated animals responded equally. The data show that chlordane differentially introduces defects in mø biochemical mechanisms associated with tumor killing.

Pulmonary macrophage antimicrobial activity in canine endotoxin shock and lung injury.

Bacterial sepsis and pneumonia are common complications of lung injury and predispose the host to a poor resolution. We studied the functional integrity of pulmonary macrophages derived from minced lung preparations in a canine model of endotoxin-induced shock with acute lung injury. Dogs given 2 mg/kg of Escherichia coli endotoxin 055:B5 developed classic shock symptoms with concomitant acute lung injury; control animals given saline showed no physiological or pathological abnormalities. Compared to previous work with this canine model, the lung injury in this extended time period (6 h) had progressed to include alveolar edema. Six hours after endotoxin infusion, the left lung was lavaged, perfused, and the resulting lung minced for isolation of pulmonary macrophages. The endotoxic-model pulmonary macrophages showed several significant functional differences from controls. Although they elicited greater production of H2O2 (p less than 0.05), both phagocytosis of radiolabeled Staphylococcus aureus and E. coli (p less than 0.05) and bactericidal activity (p less than 0.05) were diminished compared to controls. Compared to alterations previously described in alveolar macrophages, these cells produced less H2O2 and demonstrated abnormal bacterial killing at all time points. These observations suggest that the functional alterations of pulmonary macrophages that follow acute lung injury contribute to the ineffective cell-mediated antimicrobial response. These derangements may promote an increased risk of nosocomial pneumonia, the high mortality often observed subsequent to pneumonia, or the propagation of acute lung injury that facilitates respiratory failure.

Surface matrix binding alters murine peritoneal mononuclear phagocyte TNF-α and IL-6 induction

Interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS) are important promotors of mononuclear phagocyte (MO) activation. Signals derived from binding to a surface matrix also participate in promoting the activation process of MO. In this study, we examined the relative contribution of adherence in augmenting murine MO activation for cytokine production. Kinetic studies compared the production and secretion of tumor necrosis factor-alpha (TNF) and interleukin-6 (IL-6) by MO cultured as adherent monolayers to those of MO cultured as suspended cells in teflon vessels. All cells were maximally stimulated in vitro with IFN-gamma and LPS prior to analysis. Immunoprecipitation analysis of protein and RNA slot blots showed that both secreted protein and mRNA representing TNF and IL-6 are delayed two to six hours in nonadherent MO cultures compared to adherent MO cultures. Moreover, data from bioassays confirmed that these cytokines were completely functional in both systems examined. Although IFN-gamma/LPS were able to stimulate production and secretion of TNF and IL-6 in the nonadherent cells, without cell-matrix interaction, the process was significantly delayed. These data support the hypothesis that the physical event of adherence significantly facilitates the production of specific cytokines by activated MO.

Receptor-mediated ingestion responses by lung macrophages from a canine model of ARDS.

Receptor‐mediated ingestion was examined in macrophages derived from a canine model of the adult respiratory distress syndrome (ARDS). The results showed that Fc‐mediated ingestion by alveolar macrophages (AM) and macrophages from lung parenchyma (PM) was significantly diminished when compared with their respective controls. Pulsing all the experimental groups with lipopolysaccharide (LPS) for 1 hr in vitro failed to either enhance the response or return the activity to levels achieved by control cells. In parallel studies, an analysis of C3b‐mediated ingestion showed that both the experimental AM and PM performed this function only at a magnitude equal to the control cells. Similar responses were observed when an LPS pulse was performed. Although there was a reduction in Fc‐mediated ingestion and an apparent restraint of the C3b‐mediated ingestion, both AM and PM expressed a significantly enhanced ability to spread. These results suggested that the canine model of ARDS alters at least one select macrophage function that may be important to subsequently protect the host. Such disturbances in the cellular immune response may contribute to the progression of infection and lung pathology associated with this disease process.

Irradiation- and cyclophosphamide-induced alterations in Syrian hamster T-cell population activity.

The treatment of hamsters with either irradiation (IR) or cyclophosphamide (CYP) markedly alters select aspects of their cellular immune functions in a dose‐related manner. One mechanism that may be responsible for this activity appears to be the dimunition of a T‐lymphocyte subpopulation that exerts suppressive influence upon the B‐lymphocyte reactivity toward antigens. This study shows that in the hamster, immune susceptibility is affected by the magnitude and orientation of these agents (ie, IR, CYP) as they temporally relate to immunization and/or challenge with the antigen. Moreover, there is evidence that T‐independent as well as T‐dependent responses are affected by these treatments. Therefore, cyclophosphamide and irradiation modalities can be employed to modify the cellular immune responses in the hamster.

The Concurrent Expression of Griffonia simplicifolia-IB4 Binding and Tumor Necrosis Factor-α Differs between Alveolar and Peritoneal Macrophages

Abstract As a corollary to their anatomic location, alveolar macrophages (AM) have a lower threshold for generating some physiologic functions than peritoneal macrophages (PM). In this study, we examined both of these populations for their ability to bind the lectin Griffonia simplicifolia-IB4 (GSIB4) and to produce tumor necrosis factor (TNF)-α. The results showed that these two responses were concurrently expressed in activated macrophages, although they differed in magnitude when AM and PM were compared. Following in vitro incubation, AM from lipopolysaccharide-treated rats demonstrated a higher percentage of GSIB4 positivity and TNF production when compared with their respective PM. Since prostaglandin E2 can regulate the expression of some macrophage activities, experiments were conducted to determine whether this could also affect the ability of macrophages to bind the GSIB4 lectin. Neither the administration of indomethacin nor exogenous prostaglandin E2 altered the expression of this marker. Conversely, these treatments produced significant changes in TNF-α production in both alveolar and peritoneal macrophages. When the concurrent expression of GSIB4 lectin binding and TNF-α production was analyzed, AM from lipopolysaccharide-treated rats demonstrated both superior GSIB4 positivity and TNF-α production compared with all other macrophages examined. The results of this work show that AMand PMdiffer in their expression of GSIB4 binding and TNF-α production. These differential responses may be important in determining the level of activity of macrophages that are participating in an immune response.