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Hemorrhage affects the ability of murine peritoneal macrophages to alkanize intracellular pH in acidic environments
A Mayberry1, A Ayala, I H Chaudry
1Department of Surgery, Michigan State University, East Lansing 48824, USA.
Abstract:
Although departures from the normal physiologic pH range, associated with states of shock, injury, and/or infection are thought to impair phagocytic function and chemotaxis, it remains unknown if the ability of the peritoneal macrophage (pMø) to gate H+ is affected following hemorrhagic shock. To study this, male C3H/HeN mice were bled to a BP of 40 mm Hg and maintained at that level for 1 hr. Following resuscitation with the shed blood plus 2x that volume as Ringer's lactate, pMø was harvested via lavage in bicarbonate-free RPMI 1640 and enumerated to 2 million cells/ml. PMø was then loaded with a pH-sensitive dye, BCECF (2 micrograms/ml), for 20 min and washed with a buffered Na+ solution followed by phorbol myristate acetate stimulation (0.5 micrograms/ml). The capacity of the pMø to gate H+ at an external pH of 7.35 or 6.7 was assessed via fluorescence spectrophotometry. PMø from hemorrhaged animals had a decreased ability to alkalinize intracellular pH compared to that of control (P < 0.05), indicating a decreased ability of these Mø to maintain internal pH in an acidic environment following hemorrhage. In additional studies, oxidative burst capacity was determined by employing a commercially available fluorogenic substrate, (Fc Oxiburst), which fluoresces during intracellular oxidation. Fluorescence (U x 10(6)) was assessed with the ACAS 570 laser cytometer, after 0 and 200 sec following ingestion of substrate. The results demonstrate that pMø oxidative burst capacity was also decreased following hemorrhage and resuscitation.
Insights
Hemorrhagic shock impairs peritoneal macrophages' ability to regulate internal pH and reduces their oxidative burst capacity. This study investigated macrophage function following resuscitation from shock.
Area of Science:
- Immunology
- Physiology
- Cell Biology
Background:
- Physiological pH changes during shock, injury, and infection can impair immune cell function.
- The effect of hemorrhagic shock on peritoneal macrophage (pMø) pH regulation remains largely unknown.
Purpose of the Study:
- To investigate the impact of hemorrhagic shock and resuscitation on the ability of peritoneal macrophages to regulate intracellular pH.
- To assess the oxidative burst capacity of peritoneal macrophages following hemorrhagic shock.
Main Methods:
- Hemorrhagic shock was induced in mice by bleeding to a blood pressure of 40 mm Hg for 1 hour, followed by resuscitation.
- Peritoneal macrophages were isolated and loaded with a pH-sensitive dye (BCECF) to assess intracellular pH regulation.
- Oxidative burst capacity was measured using a fluorogenic substrate and laser cytometry.
Main Results:
- Peritoneal macrophages from hemorrhaged animals showed a significantly decreased ability to alkalinize intracellular pH compared to controls (P < 0.05).
- This indicates a reduced capacity of macrophages to maintain internal pH in an acidic environment post-hemorrhage.
- Oxidative burst capacity of peritoneal macrophages was also found to be decreased following hemorrhage and resuscitation.
Conclusions:
- Hemorrhagic shock and resuscitation impair the pH-gating ability of peritoneal macrophages.
- Macrophage oxidative burst capacity is compromised after hemorrhagic shock.
- These findings highlight the detrimental effects of hemorrhagic shock on critical macrophage functions, potentially impacting immune response and recovery.