Effects of extracellular pH on tumour necrosis factor-alpha production by resident alveolar macrophages.

Cellular acid-base status has been found to exert selective actions on the effector functions of activated macrophages (mphi). We examined the effects of extracellular pH (pH(o)) on the production of tumour necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in resident alveolar mphi. Cells were obtained by bronchoalveolar lavage of rabbits, activated in vitro with LPS, and cultured at pH(o) 5.5, 6.5 or 7.4 for up to 18 h. The relative abundance of TNF-alpha mRNA peaked at approximately 2 h. The peak transcript abundance was increased at lower pH(o) values. This finding probably reflected pre-transcription/transcription effects of pH, in as much as the stability of TNF-alpha mRNA induced with phorbol ester was unaffected by the experimental pH(o) values. TNF-alpha secretion by LPS-treated mphi decreased at lower pH(o) values. The TNF-alpha content of mphi-conditioned media decreased progressively with decrements in pH(o). The reduced TNF-alpha secretion at pH(o) 5.5 was accompanied by an increase in the cytosolic TNF-alpha content (compared with that at pH(o) 7.4), indicating that pH(o) altered TNF-alpha secretion due, in part, to the intracellular retention of synthesized cytokine (i.e. a post-translation effect). The data show that pH(o) has multiple effects (pre-transcription/transcription and post-translation) on TNF-alpha production induced by LPS in resident alveolar mphi. These results suggest that the role of alveolar mphi in inflammatory responses is modulated by pH(o), which may be important in tumours/abscesses and sites of infection where the external milieu is acidic.

Post-transcriptional effects of extracellular pH on tumour necrosis factor-alpha production in RAW 246.7 and J774 A.1 cells.

The present studies determined the effects of extracellular pH (pH(o)) on the production of tumour necrosis factor-alpha (TNF-alpha) in the macrophage-like cell lines RAW 246.7 and J774 A.1. The cells were activated with lipopolysaccharide (LPS) at pH(o) 5.5, 6.5 or 7.4. TNF-alpha gene transcription was monitored by Northern blot analysis. Synthesis of the cytokine was monitored by ELISA measurements of the TNF-alpha content of cell-conditioned media (extracellularly released TNF-alpha) and cell lysates (cytosolic TNF-alpha). The magnitude of the TNF-alpha response differed markedly between the two cell lines. RAW cells were more responsive to LPS than were J774 cells. However, the effects of pH(o) on TNF-alpha production were similar in the two cell lines. TNF-alpha gene transcription was insensitive to experimental pH(o). The pH(o) had no effect on the abundance of TNF-alpha mRNA at 2, 4 or 18 h. Nonetheless, synthesis of TNF-alpha was affected significantly by pH(o). The TNF-alpha contents of cell-conditioned medium and cell lysate at 18 h were reduced progressively at lower pH(o) values. The data indicate that pH(o) alters TNF-alpha production in RAW and J774 cells at a post-transcriptional level. These findings suggest that pH(o) influences the phenotypic responses of macrophages to activating stimuli and modifies the role that macrophages play in inflammatory and immune actions.

pH(i) responses to osmotic cell shrinkage in the presence of open-system buffers.

Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco(2) (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH(i)). In the present study, alveolar macrophages were exposed to hyperosmotic medium in the absence or presence of the open-system buffers CO(2)-HCO(3)(-), propionic acid-propionate, or NH(3)-NH(4)(+). In the absence of open-system buffers, exposure to twice-normal osmolarity (2T) produced a slow cellular alkalinization [change in pH(i) (DeltapH(i)) approximately 0.38; exponential time constant (tau) approximately 120 s]. In the presence of 5% CO(2), 2T caused a biphasic pH(i) response: a rapid increase (DeltapH(i) approximately 0.10, tau approximately 15 s) followed by a slower pH(i) increase. Identical rapid pH(i) increases were produced by 2T in the presence of propionic acid (20 mM). Conversely, 2T caused a rapid pH(i) decrease (DeltapH(i) approximately -0.21, tau approximately 10 s) in the presence of NH(3) (20 mM). Thus osmotic cell shrinkage caused rapid pH(i) changes of opposite direction in the presence of a weak acid buffer (contraction alkalosis with CO(2) or propionic acid) vs. a weak base buffer (contraction acidosis with NH(3)). Graded DeltapH(i) were produced by varying extracellular osmolarity in the presence of open-system buffers; osmolarity increases of as little as 5-10% produced significant DeltapH(i). The rapid pH(i) responses to 2T were insensitive to inhibitors of membrane H(+) transport (ethylisopropylamiloride and bafilomycin A(1)). The results are consistent with shrinkage-induced disequilibria in the total cellular buffer system (i.e., intrinsic buffers plus added weak acid-base buffer).

Bactericidal activity of alveolar macrophages is suppressed by V-ATPase inhibition.

Bafilomycin A(1), a selective inhibitor of V-type H(+)-translocating ATPase (V-ATPase), may be a useful adjunct in cancer chemotherapy (Altan et al. [1998] J Exp Med 187:1583-1598). Therapeutic uses of the enzyme inhibitor need to consider the agent's potential effects on normal (nontumor) cells. This study determined the effects of bafilomycin A(1) on resident alveolar macrophages (mphi). Treatment of alveolar mphi with bafilomycin A(1) (10 microM, 1 h) caused a significant decrement in cytosolic pH. This was accompanied by marked alteration of mphi bactericidal capabilities. The enzyme inhibitor caused a marginal reduction in the phagocytosis of opsonized Staphylococcus aureus and significantly suppressed intracellular killing of the phagocytosed bacteria. In keeping with the effects on intracellular killing, bafilomycin A(1) significantly reduced the production of reactive oxygen species (ROS). On the other hand, cell spreading was enhanced significantly by bafilomycin A(1). Comparable changes in ROS generation and mphi spreading were produced by altering cytosolic pH through changes in extracellular pH (pH(o)) in the absence of bafilomycin A(1). These findings suggest that the agent's effects on ROS production and mphi spreading were related to the accompanying changes in cytosolic pH. The enzyme inhibitor also altered mphi morphology, leading to the shortening of microvilli and focal loss of surface ruffles. These morphologic effects differed from those produced by altering cytosolic pH by changes in pH(o). The results demonstrate that V-ATPase activity is an important determinant of mphi functioning and structure. Therapeutic use of V-ATPase inhibitors might be expected to compromise the bactericidal activity of alveolar mphi.