Serum factor requirement for reactive oxygen intermediate release by rabbit alveolar macrophages.

Abstract

Alveolar macrophages (AM) from pathogen-free rabbits were unable to release reactive oxygen intermediates (ROI) unless they were conditioned in serum for 24-48 h before triggering with membrane-active agents. The degree of serum conditioning of AM depended upon the concentration of serum used; optimal ROI release was obtained at or above 7.5% fetal bovine serum (FBS). FBS, autologous rabbit serum, pooled rabbit serum, and pooled human serum were each capable of conditioning AM for release of ROI. Serum conditioning of AM requires synthesis of new protein(s); and the enzyme required for ROI production, NADPH oxidase, was only detectable in serum-conditioned cells. Moreover, serum-conditioned cells lost their ability to release ROI after transfer to serum-free medium, while cells maintained in serum-free medium acquired the capacity to release ROI after their transfer to serum-containing medium, demonstrating the reversibility of the phenomenon. Initial purification data indicate that conditioning is mediated by a discrete serum constituent, which precipitates 40-80% saturated ammonium sulfate, does not bind to Cibacron Blue columns, and has a molecular weight of 30,000 to 50,000, as determined by molecular exclusion chromatography. Unlike gamma interferon, which also enhances ROI release by macrophages, our serum-conditioning factor is not acid labile, retaining 67% of its activity after 120 min incubation at pH 2.0. Moreover, it does not appear to be a contaminating endotoxin, since LPS neither conditioned AM for ROI production, nor triggered ROI production by serum-conditioned AM. We propose that such a conditioning requirement may normally protect the lung against ROI-mediated tissue injury. However, during a pulmonary inflammatory reaction initiated by other mediator systems, the resulting transudation of plasma proteins into the alveolar spaces may condition AM in situ for ROI production.

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

“…Most probably the endotoxin level in FBS already exceeded the sensitivity threshold of Kupffer cells, above which they do not respond in a dose-dependent fashion to lipopolysaccharide. Similarly, Gerberick et al [10] were unable to demonstrate any enhancing action of lipopolysaccharide on the ability of serum-conditioned alveolar macrophages to produce superoxides. The addition of lipopolysaccharide to Kupffer cells from germ-free C3H mice did augment IFN production (Table 5B).…”

Cryopreserved normal macrophages as a control for assays of macrophage function

“…Although these cell lines provide the convenience of spontaneous replication and functional homogeneity, they differ from normal macrophages in several important aspects, such as expression of Ia antigen, adherence to various surfaces, and production of inflammatory cytokines (3)(4)(5)7). Such cell lines also display varying dependence on culture media supplemented with serum and additional growth factor(s) (3,13,16) which are known to have effects on normal macrophage function (1,10,11,21). Even in those cell lines that retain some membrane markers and functions characteristic of normal macrophages, subtle differences exist in their state of activation or their response to activating stimuli.…”

“…The induction and assay of 02 -release performed as reported by Gerberick (10,11) was based on the demonstration that whereas resident rabbit AM release minimal 02-when triggered in vitro with membrane-active agents, they can be conditioned in a dose-dependent manner for greatly enhanced O2-release by activating in the presence of serum or plasma for 24-48 h before triggering with phorbol myristate acetate (PMA) and measured by reduction of ferricytochrome-c in the presence of nitroprusside. IL-1 activity was measured by a thymocyte costimulation assay as described by Simon and Willoughby which was based on the uptake of [3H]thymidine in response to IL-1 stimulation (22).…”

Cotton Bract Tannin: A Novel Human T-Lymphocyte Mitogen and a Possible Causative Agent of Byssinosis

“…These lymphokines could have several in vivo effects. It has been shown that they can increase vascular permeability [23] and, therefore, cause the transudation of plasma components known to prime macrophages [24,25], or they can prime the macrophages directly [26]. It also seems that T lym phocytes are involved in the process of goblet cell metaplasia and that lymphokines stimulate goblet cell mucus production [27,28].…”