Neutrophil intracellular pH and phagocytosis after thermal trauma

Clinica Chimica Acta 295 (2000) 13–26 www.elsevier.com / locate / clinchim

Neutrophil intracellular pH and phagocytosis after thermal trauma a, a b Christopher Sachse *, Gudrun Wolterink , Norbert Pallua a

Department of Clinical Chemistry II, Medical School Hannover, Podbielskistraße 380, 30659 Hannover, Germany b Clinic of Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Hannover, Germany Received 2 August 1999; received in revised form 29 November 1999; accepted 14 December 1999

Abstract Severe burn trauma induces an acquired dysfunction of neutrophil granulocytes. As neutrophil function is considerably influenced by intracellular pH (pH i ), the pH i of blood neutrophils was longitudinally determined in 19 patients with major burns. pH i was measured by a flow cytometric method using the pH-sensitive fluoroprobe carboxy-semi-naphthorhodafluor-1; mechanisms influencing the pH i were examined by addition of amiloride (inhibition of Na 1 / H 1 countertransport), diphenylene iodonium (inhibition of NADPH oxidase) and N-formyl-methionyl-leucylphenylalanine (activation of H 1 extrusion). The neutrophil phagocytic activity was measured in parallel. Patients showed distinct alterations of neutrophil pH i , depending on whether they developed sepsis in the postburn period or not. In the sepsis patients pH i did not deviate from the values found in healthy volunteers in the first days after injury, but rose afterwards, with significant intracellular alkalinization in the second postburn week (P , 0.05). In contrast, patients without sepsis had increased pH i in the first (P , 0.01 at days 1–2), but not in the second week after burn trauma. Inhibition studies showed that postburn intracellular alkalinization is not solely caused by activation of Na 1 / H 1 countertransport. A clear relation between pH i changes and phagocytosis could not be established.  2000 Elsevier Science B.V. All rights reserved. Keywords: Burn; Neutrophil granulocyte; Intracellular pH; Phagocytosis; Flow cytometry

Abbreviations: pH i , intracellular pH; fMLP, N-formyl-methionyl-leucyl-phenylalanine; HBSS, Hanks’balanced salt solution; Hepes, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; SNARF-1-AM, SNARF-1-acetoxymethylester; DPI, diphenylene iodonium; SNARF-1, carboxy-semi-naphthorhodafluor-1 *Corresponding author. Tel.: 1 49-511-9063-357; fax: 1 49-511-9063-595. E-mail address: [email protected] (C. Sachse) 0009-8981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00189-3

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1. Introduction Infectious complications, especially sepsis, are a major cause of morbidity and death after thermal injury [1]. Multiple alterations in both the specific and nonspecific immunity have been described after burns [2–5], and although the clinical relevance of most observations remains unclear, it is widely assumed that an acquired dysfunction of the immune system predisposes the patients to severe infections, especially by bacteria. As polymorphonuclear neutrophilic granulocytes play a crucial role for protection against bacterial infections, burn-induced changes of neutrophil function have been thoroughly investigated. The alterations observed include changed expression of complement and immunoglobulin receptors [6–8], suppressed random and complement-directed migration [9–11], lowered granular content of myeloperoxidase and lactoferrin [11], altered patterns of oxygen consumption [12], diminished superoxide release and H 2 O 2 production [13–15], changed luminol-dependent chemiluminescence [16], altered phagolysosomal acidification [13,14,17] and reduced intracellular killing of bacteria and fungi [11,13–15,18]. As for neutrophil phagocytosis, there is some variability in the results reported: increased [13,19], normal [20] and lowered [11] phagocytic activity have been described after thermal injury. Differences between the methods employed with regard to opsonization probably partly account for these discrepancies [8,14]. In recent years, the effects of neutrophil intracellular pH (pH i ) on cell function have received much attention. Neutrophil chemotaxis, measured as chemotactic response to fMLP (N-formyl-methionyl-leucyl-phenylalanine), highly depends on regulation of pH i [21], and cell spreading on adhesive substrates causes a sustained cytosolic alkalinization [22]. Phagocytosis induces a rapid acidification followed by a prolonged alkalinization of neutrophil cytoplasm, the latter being caused by activation of the Na 1 / H 1 exchanger-1 [23]. Neutrophil pH i modulates the generation of superoxide radicals: intracellular alkalinization increases the amount of superoxide production and release in fMLP-stimulated cells, although a rise of pH i itself is neither necessary nor sufficient for stimulation of superoxide generation [24– 27]. However, little data is available regarding both the changes of neutrophil pH i after severe trauma, especially thermal injury, and the mechanisms underlying these changes. In the present study, we used a flow cytometric method to determine longitudinally the pH i of blood neutrophils in patients with major burns. Mechanisms influencing the pH i were examined by ex vivo inhibition and stimulation experiments. Phagocytosis was measured in parallel to investigate whether this neutrophil function is related to pH i .

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2. Materials and methods

2.1. Patients Nineteen patients (seven female, 12 male; median age, 41 years; range, 15–79) admitted to the Burn Center at Oststadtkrankenhaus, Hannover, were included in this study. The median burn size was 32% total 28 and 38 (range, 6–75%). Therapy of the patients was not influenced by their participation in this study. The patients received routine topical therapy, circulatory and nutritional support; antibiotics and ventilatory support were used when appropriate. No additional blood specimens were collected for the purposes of this study, because the samples required (approximately 1 ml of heparinized blood) could be obtained from surplus volume of specimens sent to the laboratory for routine clinical chemistry testing. The study was approved by the local Review Board. Sepsis was stated when an infectious focus (invasive burn wound infection, pneumonia, blood-borne infection) was clinically suspected or microbiologically proven and at least five of the following criteria were fulfilled for 24 h: (1) core temperature . 38.0 or , 34.08C; (2) blood leukocytes . 12 3 10 9 / l or , 4 3 10 9 / l or more than 10% promyelocytes, myelocytes and metamyelocytes in the differential leukocyte count; (3) heart rate . 90 / min; (4) blood thrombocytes , 100 3 10 9 / l; (5) systolic blood pressure , 90 mmHg; (6) therapeutic use of catecholamines; (7) serum C-reactive protein concentration increase . 30% / 24 h; (8) plasma lactate . 2.5 mmol / l; and (9) net fluid intake . 1.5 l / 24 h. Based upon these criteria patients were divided into two groups, septic and nonseptic. Both groups were compared to 20 healthy volunteers (10 female, 10 male; median age, 28 years; range, 21–44) serving as controls.

2.2. Samples Blood was examined for study at the following periods of time after thermal injury: (1) 1–2 days, (2) 3–5 days, (3) 6–8 days, (4) 9–10 days, (5) 11–12 ¨ days, and (6) 13–14 days. Heparinized tubes (Sarstedt, Nurnbrecht, Germany; final heparin concentration 12–30 IU / ml) were used for blood collection; analysis was then started within 2 h.

2.3. Intracellular pH Histopaque-1077, HBSS (Hanks’ balanced salt solution), Hepes (4-(2-hy-

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droxyethyl)-1-piperazine-ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), propidium iodide, nigericin, amiloride and fMLP were obtained from Sigma (Deisenhofen, Germany). SNARF-1-AM (carboxysemi-naphthorhodafluor-1-acetoxymethylester) was purchased from Calbiochem (Bad Soden, Germany), and DPI (diphenyleneiodonium) and dimethylformamide were from Aldrich-Chemie (Steinheim, Germany). All other chemicals were obtained from Merck (Darmstadt, Germany). Measurement of pH i followed the method described by Rothe et al. [28] with minor modification. This method uses the pH-sensitive dual-emission fluoroprobe SNARF-1 (carboxy-semi-naphthorhodafluor-1) for recording of pH i [29]. Briefly, neutrophils were prepared from 1 ml of heparinized blood by sedimentation with Histopaque-1077 for 40 min. Twenty ml of the supernatant neutrophil suspension were placed in a polypropylene reaction tube containing 1 ml standard medium (HBSS containing 10 mmol / l Hepes, adjusted to pH 7.40) incubated at 378C in a shaking waterbath. Ten ml of SNARF-1-AM working solution (20 mmol / l SNARF-1-AM and 20 ml / l dimethylformamide dissolved in standard medium) were added. Cells were incubated with SNARF-1-AM for 25 min; the reaction was then stopped by transferring the tubes to an ice-water bath. After addition of 10 ml propidium iodide solution (3 mmol / l propidium iodide, 5 mmol / l Hepes and 0.15 mol / l NaCl, adjusted to pH 7.35) the samples were kept at 48C until subjected to flow cytometric analysis. Flow cytometry was performed using a FACScan (Becton-Dickinson, San Jose, CA, USA). The laser excitation wavelength was 488 nm; orange fluorescence (490–590 nm) and red fluorescence ( . 620 nm) were measured simultaneously. Two thousand events reaching the forward scatter signal of leukocytes were analysed. Neutrophils were identified on the basis of their light scatter properties; dead cells showing high red fluorescence were excluded. The ratio of mean orange fluorescence and mean red fluorescence indicated pH i . Calibration of pH i versus fluorescence ratio was done daily by the K 1 -nigericin method [30]. With all samples pH i was determined: (1) in ‘native’ cells, as described above; (2) after addition of amiloride, an inhibitor of Na 1 / H 1 countertransport [21,31,32] (standard medium containing 1 mmol / l amiloride was used); (3) after addition of DPI, an inhibitor of the NADPH oxidase [33] (a working solution consisting of 100 mmol / l DPI and 100 ml / l dimethylsulfoxide dissolved in standard medium was made (5 ml of this solution were added together with the SNARF-1-AM working solution)); and (4) after addition of fMLP, a chemotactic peptide activating Na 1 / H 1 countertransport and other systems of H 1 extrusion [31,34] (10 ml of a fMLP working solution (10 mmol / l fMLP and 10 ml / l dimethylformamide dissolved in standard medium) were added 10 min after SNARF-1-AM).

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2.4. Extracellular pH Extracellular pH was measured as part of the routine care on a Ciba Corning 278 blood gas system (Ciba Corning, Fernwald, Germany). The instrument uses a glass electrode for pH determination; quality control was performed according to German national standards.

2.5. Phagocytosis For measurement of neutrophil phagocytosis a commercial test kit (Phagotest  , Orpegen Pharma, Heidelberg, Germany) was employed [35]. Whole blood samples were incubated with fluorescein isothiocyanate-labelled opsonized Escherichia coli for 10 min at 378C. After quenching of extracellular fluorescence and DNA staining by propidium iodide analysis was done on a FACScan flow cytometer with neutrophils being identified by their light scatter properties. The procedure determines the phagocytic activity (number of bacteria per cell) and percentage of neutrophils ingesting bacteria.

2.6. Statistical analysis As deviations from normal distribution were detected by normal probability plots, statistical analysis was performed by Mann–Whitney U-test and Spearman rank test. Median values are reported together with the mean6standard deviation. A P value less than 0.05 was regarded as significant.

3. Results

3.1. Patient groupings and outcome Nine of the 19 patients, hereafter referred to as sepsis group (median burn size, 41%; range, 29–75%), developed sepsis as defined above, usually 6–9 days after burn trauma. Three of these patients died of multiple organ failure related to sepsis. The remaining 10 patients, hereafter referred to as nonsepsis group (median burn size, 21%; range, 6–36%), had no sepsis; one patient of this group died from rupture of an aortic aneurysm. Clinical and laboratory parameters of both patient groups are summarized in Table 1.

3.2. ‘ Native’ intracellular pH For 19 burn patients, neutrophil ‘native’ pH i did not differ significantly from

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Table 1 Clinical and laboratory parameters of septic and nonseptic burn patients a Parameter

Sepsis group (n 5 9)

Nonsepsis group (n 5 10)

Core temperature (8C, highest individual values) Leukocyte count ( 3 10 9 / l, highest individual values) Heart rate (1 / min, highest individual values) Platelet count ( 3 10 9 / l, lowest individual values) Number of patients with hypotensive episode (systolic blood pressure , 90 mmHg) Number of patients with catecholamine therapy Serum C-reactive protein concentration (mg / l, highest individual values) Plasma lactate concentration (mmol / l, highest individual values) Net fluid intake (l / 24 h, highest individual values)

39.460.9

38.960.7

32.969.3

22.9610.8

15967

120627

54631

123638

9

4

9 377682

6 2266138

5.662.8

2.561.5

12.265.0

4.562.7

a

Data are given as mean6standard deviation unless otherwise stated.

that of 20 healthy volunteers (7.3660.16; median, 7.40) from days 1–14 after thermal injury. However, separate analysis of the sepsis and nonsepsis groups showed distinct patterns of pH i deviations. In the sepsis group neutrophil pH i was essentially normal 1–2 days after burn trauma (7.3160.14; median, 7.31), but increased afterwards, with pH i values significantly elevated versus controls in the second postburn week (Fig. 1, open columns). In contrast, significantly increased pH i values were observed in the nonsepsis group during the first postburn week (days 1–2, 7.5660.16; median, 7.58; P , 0.01), with normalization afterwards (Fig. 2, open columns).

3.3. Intracellular pH — inhibition and stimulation experiments After addition of amiloride, controls’ neutrophil pH i fell to 6.9060.36 (median, 6.93). Amiloride caused intracellular acidification, too, in neutrophils of burn patients, both in the sepsis and nonsepsis group. Amiloride-induced changes of pH i were similar in all groups; therefore, differences between patient groups and controls observed with ‘native’ cells remained largely unchanged in numerical values. However, inhibition of Na 1 / H 1 countertransport by amiloride

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Fig. 1. Intracellular pH in ‘native’ (open columns) and fMLP-stimulated (closed columns) neutrophils of sepsis group burn patients. The values are given as mean6standard deviation. Solid and dotted lines indicate the mean6standard deviation region of intracellular pH in ‘native’ and fMLP-stimulated neutrophils of controls, respectively. The significances of differences between sepsis group patients and controls are indicated as *P , 0.05.

increased intra-group variability, so that statistical significance was not reached (data not shown in detail). Inhibition of the NADPH oxidase by DPI had little influence on neutrophil pH i of controls (7.4460.18; median, 7.47) and of both septic and nonseptic patients. Again, there was a tendency towards increased intra-group variability after addition of DPI, and differences between patient groups and controls generally became not significant (data not shown in detail). In vitro stimulation by fMLP resulted in a marked rise of controls’ neutrophil pH i (7.7660.22; median, 7.79). fMLP-induced intracellular alkalinization was observed, too, in patients’ neutrophils; however, its degree depended on the pH i values of the ‘native’ cells. If pH i was increased versus controls in ‘native’ cells (throughout the second postburn week in the sepsis group and throughout the first postburn week in the nonsepsis group), further fMLP-induced alkalinization was reduced, so that pH i values of fMLP-stimulated neutrophils generally did not exceed those of the control group (Figs. 1 and 2).

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Fig. 2. Intracellular pH in ‘native’ (open columns) and fMLP-stimulated (closed columns) neutrophils of nonsepsis group burn patients. The values are given as mean6standard deviation. Solid and dotted lines indicate the mean6standard deviation region of intracellular pH in ‘native’ and fMLP-stimulated neutrophils of controls, respectively. The significances of differences between nonsepsis group patients and controls are indicated as *P , 0.05, **P , 0.01.

3.4. Relation between intracellular and extracellular pH Correlation analysis did not reveal a relation between individual intracellular and extracellular pH values for any time period (data not shown).

3.5. Phagocytosis In healthy volunteers the phagocytic activity (number of bacteria per cell, expressed as mean channel number of fluorescence) and the percentage of phagocytic neutrophils were 802644 (median, 803) and 89.266.3% (median, 90.5%), respectively. For the whole group of 19 burn patients, significantly different values for neutrophil phagocytosis were not measured in our study. However, the sepsis group patients showed reduced phagocytic activity, with significant differences versus controls from days 1 to 10 after trauma (Fig. 3, open columns). Additionally, the percentage of phagocytic neutrophils was

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Fig. 3. Phagocytic activity (number of bacteria per cell, expressed as mean channel number of fluorescence) in sepsis group (open columns) and nonsepsis group (closed columns) burn patients. The values are given as mean6standard deviation. Solid lines indicate the mean6standard deviation region of phagocytic activity in neutrophils of controls. The significances of differences between patients and controls are indicated as *P , 0.05, **P , 0.01.

significantly diminished 1–2 days after burn injury (76.8612.4%; median, 72.6%; P , 0.01). In the nonsepsis group phagocytic activity and percentage of phagocytic neutrophils generally were normal; a significantly decreased phagocytic activity was recorded only for the 6–8 days time period (Fig. 3, closed columns).

3.6. Relation between intracellular pH and phagocytosis To examine whether phagocytosis is related to ‘native’ pH i on an individual basis, correlation studies were done separately for all time periods. No significant correlation could be found by Spearmans rank test; however, at days 1–2 there was a tendency towards a more pronounced phagocytosis in patients with a more alkaline neutrophil pH i (r S 5 0.39, P 5 0.10).

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4. Discussion Bacterial sepsis and other infectious complications occur frequently after thermal injury and are probably related to burn-induced alterations which have been described with regard to many aspects of host defense. In particular, neutrophil function is impaired after burn trauma. Neutrophil immunocompetence depends on intact regulation of intracellular pH, and mechanisms influencing pH i have been thoroughly investigated in recent years. The bactericidal function of neutrophils is effected to a large extent by the respiratory burst, a sequence of reactions transforming molecular oxygen into toxic products. These reactions are initiated by the NADPH oxidase and associated with a considerable rise of intracellular acid generation [36]. As many cell functions depend on the maintenance of pH i within relatively narrow ranges, the excess acid generation must be compensated to preserve cell viability and microbicidal responses [25]. One mechanism of H 1 extrusion, the Na 1 / H 1 countertransport system, has now been described for more than 10 years [31,37]. This system executes a 1:1 Na 1 / H 1 exchange; it is functional in quiescent neutrophils and can be further activated by exposure to fMLP or phorbol-12myristate-13-acetate, with the activation being mediated by protein kinase C and possibly by other mechanisms [24,38,39]. Phagocytosis, too, induces a rise of neutrophil Na 1 / H 1 exchange [23]. Identification of Na 1 / H 1 countertransport activity is facilitated by its high sensitivity to amiloride and its analogues [31,32]. More recently, two other systems effecting H 1 extrusion in neutrophils have been found: a electrogenic vacuolar-type H 1 -ATPase (V-pump) and a purely passive H 1 conductance. Both systems show no activity in quiescent neutrophils, but can be stimulated by exposure to fMLP or phorbol esters [34,39–41]. To date, it is not clear to what extent these mechanisms contribute to regulation of pH i under physiological and pathological conditions. Our results show that disturbances of pH i regulation can be observed in blood neutrophils after severe thermal injury and that these changes are related to the clinical course of the patients. Patients of the sepsis and nonsepsis groups showed distinct, characteristic alterations of neutrophil pH i . Since many stimuli such as fMLP, phorbol-12-myristate-13-acetate or zymosan, which probably imitate the trauma-induced activation, cause a prolonged intracellular alkalinization of neutrophils [23,24,31,38], we expected an increase of cell pH i in the first days after thermal injury. However, this early intracellular alkalinization could only be observed in the nonsepsis group. In contrast, patients of the sepsis group generally did not show a rise of neutrophil pH i before the second postburn week, when imminent or overt infection may contribute to blood neutrophil stimulation. We only can speculate as to the causes of the different neutrophil pH i time

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courses in the sepsis and nonsepsis groups. It should be noted that average burn sizes differed considerably between the two groups; more extended trauma may result in a higher rate of extravasation of activated neutrophils, so that the discrepancies observed in the peripheral blood just may represent selection effects. Additionally, the correlation between burn size and granulocyte left shift has to be taken into consideration: pH i regulation could be different in immature neutrophils. However, it can be expected that the majority of immature granulocytes was excluded from flow cytometric analysis because of their altered light scatter properties. Finally, there is the possibility that overstimulation of neutrophils inhibits the pH i responses. Anti-inflammatory cytokines or prostaglandins could be involved in this inhibitory pathway. After inhibition of the Na 1 / H 1 countertransport system with amiloride intracellular acidification was similar in burn patients’ and controls’ neutrophils, with differences observed with ‘native’ cells remaining quite unchanged. This finding came as a surprise as it is now generally accepted that stimuli like FMLP, phorbol-12-myristate-13-acetate or zymosan cause intracellular alkalinization exclusively via activation of Na 1 / H 1 exchangers, which can be inhibited by amiloride [23,31,32,38]. Therefore we expected the alkalinization of burn patients’ neutrophils to disappear after incubation with amiloride. Our findings suggest that mechanisms other than activation of the Na 1 / H 1 countertransport system contribute to the increase of pH i after thermal injury. NADPH oxidase inhibition by DPI did not have major influence on neutrophil pH i , showing that either spontaneous H 1 generation by blood neutrophils is low or that it is well compensated. As expected, further fMLP-induced alkalinization was diminished in prestimulated, already alkalized cells. Regarding phagocytosis our results confirm reports of reduced neutrophil phagocytic activity [11], especially in the patients with more extended burn trauma. As opsonized bacteria were employed for measurement of phagocytosis, this reduction is independent of postburn decrease of serum opsonins. However, we were not able to establish a clear link between neutrophil pH i regulation and phagocytosis. In conclusion, regular patterns of neutrophil pH i changes can be observed after burn trauma. These patterns differ in relation to the clinical outcome of the patients, although we only can speculate whether these differences are caused by the development of infection itself or by discrepancies regarding the trauma size. However, neutrophil pH i changes constitute an interesting object of investigation. Our data suggest that mechanisms other than the Na 1 / H 1 countertransport system — such as the electrogenic vacuolar-type H 1 -ATPase or the passive H 1 conductance — contribute to the disturbances of pH i regulation after thermal injury. Experiments using specific inhibitors of these mechanisms could provide further insight into the role of pH i changes for neutrophil function.

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Acknowledgements The study was supported by a research grant of the ‘Gesellschaft der Freunde der Medizinischen Hochschule Hannover’.

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