INFECrION AND IMMUNITY, July 1992, P. 2957-2968

Vol. 60, No. 7

0019-9567/92/072957-12$02.00/0 Copyright X) 1992, American Society for Microbiology

Propionate Induces Polymorphonuclear Leukocyte Activation and Inhibits Formylmethionyl-Leucyl-PhenylalanineStimulated Activation BEATRICE A. BRUNKHORST,t ELI KRAUS, MADALENNA COPPI,4 MICHAEL BUDNICK, AND RICHARD NIEDERMAN* Department of Cell Biology, Forsyth Research Institute, 140 The Fenway, Boston, Massachusetts 02115 Received 14 January 1992/Accepted 1 May 1992

Short-chain carboxylic acids (SCCA) are metabolic by-products of bacterial pathogens which can alter cytoplasmic pH and inhibit a variety of polymorphonuclear leukocyte (PMN) motile functions. Since cytoskeletal F-actin alterations are central to PMN mobility, in this study we examined the effects of SCCA on cytoskeletal F-actin. Initially, we tested nine SCCA (formate, acetate, propionate, butyrate, valerate, caproate, lactate, succinate, and isobutyrate). We document here that while eight altered cytoplasmic pH, only six altered cytoskeletal F-actin. We then selected one SCCA that altered both F-actin and cytoplasmic pH (propionate) and one SCCA that altered only cytoplasmic pH (lactate) for further study. Propionate, but not lactate, caused an irregular cell shape and F-actin distribution. Furthermore, propionate, but not lactate, inhibited formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated PMN polarization, F-actin localization, and cytoplasmic pH oscillation. Propionate-induced changes in cytoskeletal F-actin and cytoplasmic acidification were not affected by the fMLP receptor antagonist N-t-BOC-1-methionyl-l-leucyl-l-phenylalanine; however, alkalinization was affected. Pertussis toxin treatment completely inhibited propionate-induced changes in F-actin but had no effect on propionate-induced cytoplasmic pH oscillation. These results indicate that propionate (i) bypasses the fMLP receptor and G protein(s) to induce cytoplasmic pH oscillation, (ii) operates through G protein(s) to induce actin oscillation, cell shape changes (to irregular), and F-actin localization, and (iii) inhibits fMLP-stimulated cytoplasmic pH and actin oscillation, PMN polarization, and F-actin localization.

Neutrophils provide the first line of host defense against bacterial pathogens. How and why the delicate balance between the polymorphonuclear leukocytes (PMNs) and bacteria is swayed toward the bacteria in disease is often not clear. It is clear that chemical mediators which activate or inactivate the PMNs initially interact with receptors, secondarily activate biochemical responses, and finally activate cellular responses (5, 37, 50). Bacterial mediators found at the sites of infection may use similar pathways to activate or inactivate PMN function (1, 10, 11, 25, 43, 59). Short-chain carboxylic acids (SCCA) are metabolic byproducts from pathogenic anaerobic bacteria and appear in millimolar concentrations at the sites of infection (1, 6, 17, 28, 51, 58). Studies from several laboratories indicate that SCCA inhibit stimulated PMN functions, such as chemotaxis, phagocytosis, degranulation, oxidative burst, and phagocytic killing of ingested bacteria (9, 4244, 55, 59). Despite considerable evidence regarding the effects of SCCA on PMNs, the mechanism(s) of SCCA-induced inhibition is unknown. Paradoxically, some SCCA can stimulate cytoskeletal F-actin polymerization, right-angle light scatter, and changes in cytoplasmic pH (25, 54, 59) yet inhibit motile functions (1). The purpose of this study was to examine the mechanism of SCCA-mediated PMN inhibition. Since some SCCA are commonly used to experimentally alter pH (4) and can therefore potentially alter motile functions, we examined the effect of SCCA on cytoplasmic pH. Furthermore, since some SCCA inhibit PMN motility and PMN motility is

dependent on actin oscillation (20, 37), we examined the effects of SCCA on cytoskeletal F-actin oscillation, localization, and cell shape. Finally, we examined the effects of selected SCCA (propionate and lactate) on formylmethionylleucyl-phenylalanine (fMLP)-stimulated responses, interaction with fMLP receptor, and G proteins. The results suggest that SCCA may inhibit PMN motility by altering fMLPstimulated oscillation of pH, actin, and cell shape. MATERIALS AND METHODS

Materials. Nitrobenzo-oxadiazole phallacidin (NBD-phallacidin) and 2',7'-bis-(2-carboxy-ethyl)-5-(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) were obtained from Molecular Probes, Inc. (Eugene, Oreg.). Dimethyl sulfoxide, ethylene glycol-bis(13-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), fMLP, N-t-BOC-1-methionyl-1-leucyl-1phenylalanine (tBOC-fMLP), N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (HEPES), MgCl, piperazine-N,N'bis(2-ethanesulfonic acid) (PIPES), Triton X-165, n-propyl gallate, nigericin, sodium propionate, sodium DL-lactate, sodium succinate, sodium butyrate, sodium isobutyrate, sodium caproate, sodium formate, and acetic acid (free acid) were from Sigma Chemical Co. (St. Louis, Mo.). Ficoll-Hypaque and Mono-Poly Resolving Medium were from Flow Laboratories (McLean, Va.). Hanks balanced salt solution (HBSS), with and without Ca2 + and Mg2+, and 3-(N-morpholino)propanesulfonic acid (MOPS) were from GIBCO-BRL (New York, N.Y.). Calibrated fluorescent polystyrene beads were from Flow Cytometry Standards Corp. (Research Triangle Park, N.C.). Pertussis toxin (PT) was from List Biological (Campbell, Calif.).

* Corresponding author. t Present address: Dana Farber Cancer Institute, Boston, MA

02115. t Present address: Harvard

Stimulators. fMLP and phorbol myristate acetate (PMA)

University, Cambridge, MA 02138. 2957

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BRUNKHORST ET AL.

were dissolved in dimethyl sulfoxide and diluted in HBSS plus 10 mM HEPES (pH 7.4). All SCCA were diluted in HBSS plus 10 mM HEPES and adjusted to pH 7.4, using NaOH or HCl. Isolation of neutrophils. Whole blood was drawn from healthy donors by venipuncture into Vacutainers containing 1,500 U of heparin per ml. Platelets were separated by centrifugation at 200 x g at room temperature for 12 min. The platelet-rich plasma was aspirated, and the remaining cell layer was diluted in Ca2+-and-Mg2+-free HBSS-10 mM HEPES buffer (pH 7.4) (CMF-HBSS+HEPES) to the original volume. The leukocytes were separated by centrifugation as previously described (2, 23). All assays were performed with neutrophils diluted in complete HBSS buffer. NBD-phallacidin staining of neutrophils. Actin polymerization was measured using the NBD-phallacidin method essentially as described previously (19). The initial experiments shown in Fig. 1 were done at 25°C, and all subsequent ones were performed at 37°C. Neutrophils were warmed to 37°C for 5 min before stimulation. At timed intervals under the conditions described in the figure legends, 200-,ul samples of the cell suspension were removed and simultaneously fixed and extracted at 4°C, as described previously (2). Microscopic examination of NBD-phallacidin-stained cells. Cells fixed and stained for F-actin as described above were sedimented on glass slides using a Shandon-Eliot cytocentrifuge. Cells were then rehydrated in a solution of 90% glycerol, lx PF-PHEM, and 2.5% n-propyl gallate (to prevent photobleaching) (14). Visual examination was done and pictures were taken on a Nikon inverted light microscope using phase-contrast or fluorescence optics. Pictures were taken with Fujichrome 1600 and shot at ASA 400 and pushed once in development. Identifying marks on slides were covered, and slides were counted without knowledge of incubation conditions. All cells in 10 fields on each slide were counted for a total of > 100 cells per slide. Two photographs of typical fields were taken for each cell stimulation. The cell shape and F-actin localization were determined and put into three categories, round, polar, and irregular, in a manner similar to that described by MacFarlane et al. (31). We define here the round category as those cells that appeared round with uniform F-actin staining, typically displayed by unstimulated cells. In contrast, cells stimulated with fMLP were elongated and had a bipolar shape with F-actin localized to the cell anterior (defined as polar). Finally, cells exposed to propionate had a cell shape that was irregular with two or more F-actin localizations (defined as irregular). Cytometric analysis of NBD-phallacidin-stained cells. Fluorescence was measured on a Coulter EPICS C flow cytometer equipped with a Coulter MDADS data acquisition system, as previously described (2, 19, 25). Polymerization and depolymerization rates were calculated as the relative change in F-actin per minute. Cytoplasmic pH determination. PMNs in CMFHBSS+HEPES at 106/ml were loaded with a 4 ,uM final concentration of the cytoplasmic pH-sensitive dye BCECF-AM for 30 min at 37°C. Residual BCECF-AM was washed from the cells by the addition of three times the original volume of CMF-HBSS+HEPES and centrifuged at 200 x g at 4°C for 10 min. The pellet was brought to 106 PMNs per ml in HBSS plus 10 mM HEPES and kept on ice until fluorometric measurements were performed. Fluorescence was measured in an SLM-500C spectrofluorometer with a thermostat set at 37°C and equipped with a stirring mechanism, injection port, and IBM data acquisition sys-

INFECT. IMMUN.

tem. Initial experiments shown in Fig. 3 were performed at 25°C, with all subsequent ones, including those with HCl, performed at 37°C. The BCECF-loaded cells were excited at 450 and 500 nm, and emissions were collected at 530 nm. The changes in BCECF fluorescence ratio [ratio = (emission at 530 nm/excitation at 500 nm) - (emission at 530 nm/excitation at 450 nm)] were correlated to changes in cytoplasmic pH with a standard curve created by the addition of the K+/H+ ionophore nigericin (10 mM) to suspensions of cells in the high-potassium MOPS buffer with four different pHs (6.5, 6.9, 7.3, and 7.7) (54). The baseline pH, measured for 30 s before the stimulus was added, was 7.0 + 0.1. The relative pH is the pH value normalized against this baseline reading. Stirred cells in the spectrofluorometer cuvette typically displayed a decrease in the fluorescence ratio during the assay as a result of leakage of the dye. Therefore, the fluorescence readings of stirred cells during the assay were collected for each experiment, and the absolute value of the ratio was added to the value for each subsequent cytoplasmic pH oscillation. tBOC-fMLP treatment of neutrophils. Changes in cytoskeletal F-actin and cytoplasmic pH were measured in neutrophils (106/ml in HBSS plus 10 mM HEPES) incubated in buffer or 10 ,uM tBOC-fMLP for 5 min followed by stimulation. PT treatment of neutrophils. PMNs (106/ml) were preincubated with buffer or 500 ng of PT for 2 h at 37°C as previously described (2). Cells were washed in CMF-HBSS+HEPES and resuspended in cold HBSS plus 10 mM HEPES. PT treatment did not alter the baseline F-actin content or cytoplasmic pH (data not shown). All results are expressed as the percentages of responses in cells preincubated with buffer. Statistics. All data are reported as means + standard errors of the means. Statistical significance between the means was assessed by the Student t test.

RESULTS Effect of SCCA on cytoskeletal F-actin and cytoplasmic pH. Previous studies indicated that SCCA inhibited PMN motile functions. Since alterations in cytoskeletal F-actin are thought to affect cell motility, our initial studies investigated which SCCA had the most effect on cytoskeletal F-actin. The SCCA used were chosen because they are produced by pathogenic bacteria (1, 6, 28, 51). Figure 1 displays the effects of SCCA on PMN F-actin. SCCA were added in buffered HBSS (pH 7.4) to more closely approximate the in vivo environment. We chose concentrations of SCCA that were significantly above the normal level in serum of 1 nM (7). The linear SCCA are arranged in order of increasing carbon chain length, with the SCCA with carbon branches or substitutions shown last. SCCA induced the maximal F-actin response, which increased with increasing linear carbon chain length to three carbons and then decreased with increasing carbon chain length. Propionate and acetate induced the highest F-actin peak. However, the ability to induce increases in F-actin is not solely dependent on the length of the carbon chain. For example, propionate with three carbons generated a response similar to that of fMLP (relative F-actin maximum of 2.15 + 0.03), while lactate induced no response. Taken together, these results indicate that six of the nine SCCA tested induced a significant increase in F-actin. Figure 2 provides a typical time course of the propionateinduced F-actin oscillation. It indicates that propionate

PROPIONATE ACTIVATES PMNs YET INHIBITS fMLP STIMULATION

VOL. 60, 1992

2959

1

Relative F-Actin

2

L 2L1 ~ ~ 12 1.02

Formate

|

Propionate

1.161. | l 1.06 1.01 n1.01

Valerate

Lactate

Isobutyrate

5 mM

Acetate Butyrate Caproate Succinate FIG. 1. Effect of SCCA on cytoskeletal F-actin. The maximum cytoskeletal F-actin contents of PMNs were determined after exposure to 5, 10, and 30 mM SCCA. In all cases, maximum F-actin levels occurred between 10 and 45 s after stimulation. The highest F-actin peak values were compared with buffer control values, using Student's t test. Values are mean F-actin peaks induced by each SCCA and are from at least three experiments performed at room temperature with different blood donors. Values statistically different from the values for unstimulated cells are indicated by asterisks (** indicates P < 0.001; * indicates P < 0.02). The data indicate that acetate, propionate, butyrate, caproate, and isobutyrate, and fMLP, but not formate, lactate, or succinate, caused changes in cytoskeletal F-actin content.

induced a rapid polymerization in cytoskeletal F-actin within 10 s, followed by depolymerization to a level above the baseline level. In fact, butyric acid and acetate also caused a transient increase in F-actin content (data not shown). It should be noted that the limit of resolution is 10 s. Therefore, the peak may occur earlier and may be higher. Given this limitation, the SCCA with the fastest polymerization rate also had the fastest depolymerization rate. For example, propionate, butyrate, and acetate have polymerization rates of 3.59 + 0.09, 1.63 + 0.20, and 1.06 + 0.03 and depolymerization rates of 2.97 + 0.09, 1.51 + 0.20, and 1.34 + 0.03 (percent change per minute), respectively. In contrast, 30 mM lactate had no significant effect on F-actin levels during the 5 min of measurement. Furthermore, propionate, but not lactate, induced an actin oscillation that was similar in magnitude to that induced by fMLP (Fig. 2). Previous reports from this and other laboratories suggest that intracellular pH changes may affect alterations in cytoskeletal F-actin (11, 33, 36, 48, 55). Furthermore, for over 50 years, SCCA have been used to alter intracellular pH (4, 9). Therefore, the ability of SCCA to alter PMN intracellular pH was determined. Figure 3 displays the maximum changes in acidification and alkalinization induced by the SCCA tested. These results indicated that all SCCA caused an acidification and a subsequent increase in cytoplasmic pH. Valerate- and

caproate-induced acidification did not return the pH values to baseline levels. They remained below baseline values, at -0.25 and -0.1 pH unit, respectively. However, formate, acetate, propionate, and lactate induced an increase in cytoplasmic pH above the baseline value. HCI, on the other hand, caused an acidification that was constant throughout the 5 min of measurement. Taken together, these results indicate that all seven SCCA tested caused intracellular acidification and alkalinization. Kinetic studies indicated that propionate caused rapid acidification followed by alkalinization that were similar to those elicited by fMLP in both magnitude and timing (Fig. 4). Notice that while lactate caused the most initial acidification, propionate induced alkalinization faster. These kinetic studies indicated that fMLP induced rapid acidification followed immediately by alkalinization (Fig. 4). Therefore, these results indicate that propionate, but not lactate, induced a cytoplasmic pH oscillation similar to that elicited by fMLP. Effect of propionate and lactate on fMLP-stimulated PMN responses. The above results indicated that propionate and fMLP, but not lactate, can alter both cytoskeletal F-actin and pH to similar extents. Therefore, we next determined whether preincubation with propionate could affect fMLP-induced cell shape and F-actin localization. We chose lactate as the control

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INFECT. IMMUN. 0.20

- fMLP 0.15 2.0 _

Lactate

0.10

z

0.05 w

>

w

1.5

0.00 -0.05 -0.10 -0.15

0

1

2 3 4 5 TIME (min) FIG. 2. Representative time courses showing the effect of propionate, lactate, and fMLP on cytoskeletal F-actin. Cells were exposed to 5 mM propionate, 30 mM lactate, or 10-8 M fMLP at time zero. Data are representative of at least five experiments performed at 37°C on different blood donors. The data shown indicate that propionate, but not lactate, causes changes in cytoskeletal F-actin that are similar to those elicited by fMLP.

2 3 4 5 TIME (min) FIG. 4. Time courses representing changes in cytoplasmic pH induced by propionate, lactate, and fMLP. Cells were exposed to 5 mM propionate, 30 mM lactate, or 10-8 M fMLP at time zero. Data are representative of at least five experiments performed on different blood donors. The data indicate that both propionate and lactate cause changes in cytoplasmic pH. The change in cytoplasmic pH induced by propionate is similar to that caused by fMLP.

for these experiments because although it did not affect F-actin, its carbon chain length is similar to that of propionate. The photomicrograph shown in Fig. 5A displays a typical unstimulated PMN. It is round and has a uniform cytoskel-

etal F-actin distribution. fMLP stimulated a polarized cell shape and F-actin localization to the cell anterior (Fig. 5B). Cells incubated with propionate display an irregular shape (Fig. 5C), whereas cells incubated with lactate remain round (Fig. SD). In contrast, cells that had been preincubated in propionate and then exposed to fMLP displayed an irregular shape and two or more areas of F-actin localization (Fig. SE). The statistical data presented in Fig. 6 show that the cells in the photomicrographs represent the principal shapes and F-actin localizations displayed by cells in the three conditions indicated. For example, fMLP caused the majority of the cells preincubated in buffer or lactate to display a polar cell morphology and polar F-actin distribution (fMLP, 93% + 3% polar; lactate plus fMLP, 84% + 1% polar). In contrast, only 12% + 3% of the cells preincubated with propionate and then stimulated with fMLP exhibited a polar shape and polar F-actin distribution. In fact, the majority (61% 10%) of the cells preincubated with propionate and stimulated with fMLP had an irregular shape and irregular F-actin distribution. Propionate alone also caused the majority of the cells to exhibit an irregular shape and F-actin localization (percent round = 35 15; percent irregular = 53 + 16) (Fig. 5C), while buffer or lactate alone had no significant effect (percent round = 89 5 and 96 + 1, respectively) (Fig. 5A and D). Therefore, propionate, but not lactate, altered the PMN shape and cytoskeletal F-actin localization and altered the fMLP-stimulated shape change and cytoskeletal F-actin localization. These results lead us to question whether irregular shapes represented different cell populations with different F-actin contents. Figure 7A, B, and C displays the flow cytometric log fluorescence histograms exhibited by unstimulated cells and cells incubated with lactate or propionate (unshaded histograms) and then exposed to fMLP (shaded histograms), respectively. The unstimulated cells displayed an F-actin content with a normal cell distribution (unshaded histogram in

0

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0.1

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z

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-za Alkalinizatior

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STIMULANT FIG. 3. Effect of SCCA on cytoplasmic pH. Cells were exposed to 10 mM (each) SCCA, 10 nM fMLP, or 10 ,uM HCI. In all cases, maximum acidification occurred within 10 to 45 s after the addition of SCCA. Values are the means ± standard errors of the means of maximum changes in pH of at least three experiments performed on different blood donors. The data indicate that all SCCA tested, fMLP, and HCl cause changes in cytoplasmic pH. Abbreviations: For, formate; Ace, acetate; Pro, propionate; But, butyrate; Val, valerate; Cap, caproate; Lac, lactate.

PROPIONATE ACTIVATES PMNs YET INHIBITS fMLP STIMULATION

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FIG. 5. Fluorescence photomicrographs of PMNs demonstrating the effects of SCCA on cytoskeletal F-actin localization and cell shape. Representative photomicrographs of PMN stained for F-actin were taken either at time zero (A), 5 min after the addition of 10 nM fMLP to cells preincubated in buffer for 15 min (B), 5 min after the addition of 5 mM propionate to cells preincubated in buffer for 15 min (C), 5 min after the addition of 30 mM lactate to cells preincubated in buffer for 15 min (D), and 5 min after the addition of 10 nM fMLP to cells preincubated in 5 mM propionate for 15 min (E). These five photomicrographs are representative of the three categories for cell shape and F-actin localizations found: round (A and D), polar (B), and irregular (C and E). See Materials and Methods for definitions of cell shape and localization categories. These micrographs indicate that propionate alters fMLP-induced cell shape and F-actin localization.

Fig. 7A). Similarly, cells incubated in propionate or lactate also displayed a normal distribution and level of F-actin content. Stimulation with 10 nM fMLP caused 95% + 2% of the cells treated with buffer, lactate, or propionate to display a maximal increase in log F-actin content within 10 s (shaded histograms in Fig. 7A, 13, and C, respectively). Therefore, these results demonstrate that the majority of cells treated in all conditions displayed a homogeneous F-actin response, despite cell shape and F-actin localization differences. The above data indicate that microscopic analysis can identify important changes in cell shape and F-actin localization that would be missed by flow cytometry assessment of F-actin content. We therefore more closely examined differences in microscopic versus flow cytometric analysis of F-actin. Cells preincubated for 15 min in buffer, 5 mM propionate, or 30 mM lactate displayed similar F-actin levels (1.04 + 0.19 for buffer, 0.85 ± 0.05 for lactate, and 1.21 ±

0.05 for propionate). These data suggest that propionate had no significant effect on F-actin content by the end of the 15-min incubation. Therefore, these data are in direct contrast to data showing that propionate alone causes a irregular cell shape and F-actin distribution (Fig. SC). In addition, flow cytometric F-actin content analysis indicated that cells preincubated in buffer, lactate, or propionate and then stimulated with fMLP for 5 min had statistically similar values (1.6 ± 0.2 for buffer, 1.25 ± 0.11 for lactate, and 1.9 ± 0.1 for propionate). However, the photomicrographs in Fig. 5 demonstrate that propionate completely changes the normal F-actin distribution and cell shape elicited by fMLP. Therefore, these findings further highlight the effect of SCCA on PMN. Kinetic studies revealed that cells treated in buffer, lacA

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Ikm LMIi -M1 br m AN 1 1MVI lAM I u Li1 .14 ROUND POLAR IRREGULAR CELL SHAPE AND F-ACTIN LOCALIZATION FIG. 6. Effect of SCCA on stimulated cell shape and cytoskeletal F-actin localization. PMNs were incubated for 15 min with buffer, 30 mM lactate, or 5 mM propionate and stimulated with 10 nM fMLP for 5 min. Values are means standard errors of the means for three experiments performed on different blood donors. Values statistically (P < 0.02) different from the values for cells preincubated for 15 min with buffer and then stimulated with 10 nM fMLP are indicated (*). The data indicate that propionate, but not lactate, alters fMLP-induced cell shape and F-actin localization.

0

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-q

LOG F-ACTIN CONTENT FIG. 7. Representative histograms showing log F-actin fluorescence readings of PMNs. The unfilled histograms represent the distribution of PMNs treated with buffer (A), 30 mM lactate (B), or 5 mM propionate (C) for 15 min. Hatched histograms are representative of log F-actin fluorescence after exposure to 10 nM fMLP for 10 s (time of maximal F-actin) after 15-min preincubation with buffer, lactate, or propionate. Each histogram represents >95% of the cell population from that respective cell sample. Data are representative of at least three experiments performed on different blood donors. The data indicate that propionate and lactate have no effect on the population homogeneity of the F-actin response.

2962

BRUNKHORST ET AL.

INFECT. IMMUN.

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FIG. 8. Representative time courses displaying the effects of SCCA on stimulated F-actin. Cells were incubated for 15 min in buffer, 30 mM lactate, or 5 mM propionate and exposed to 10 nM fMLP at time zero. Data are representative of three experiments performed on different blood donors. This datum indicates that propionate, but not lactate, increases the fMLP-induced peak F-actin. tate, or propionate and then stimulated with fMLP exhibited

rapid F-actin polymerization followed by depolymerization and eventual stabilization in F-actin content (Fig. 8). However, propionate preincubation significantly increased the initial F-actin peak induced by fMLP compared with cells preincubated in buffer or lactate (2.21 0.12 for buffer, 2.25 0.18 for 30 mM lactate, and 2.73 0.16 for 5 mM propionate; P < 0.05) but had no significant effect on the depolarization rate. These results indicate that propionate, but not lactate, altered the fMLP-stimulated F-actin peak. Taken together, these results suggest that propionate, but not lactate, (i) caused irregular cell shape and F-actin localization when added alone or prior to the fMLP addition, (ii) increased the fMLP-elicited F-actin polymerization, yet had no effect on the fMLP subsequent depolymerization, and (iii) had no effect on the F-actin content S min after the addition of fMLP. Given the effects of propionate and lactate on fMLPstimulated F-actin and cell shape, we next investigated the effects of propionate and lactate on fMLP-stimulated cytoplasmic pH changes. The summary data provided in Table 1 indicate that fMLP caused rapid acidification in cells preincubated with buffer, lactate, or propionate. However, propionate inhibited fMLP-induced alkalinization. It should be a

±

±

±

Treatment

1

2 3 4 5 TIME (min) FIG. 9. Representative time courses displaying the effects of SCCA on cytoplasmic pH. Cells were incubated for 15 min with buffer, 30 mM lactate, or 5 mM propionate and exposed to 10 nM fMLP at time zero. Data are representative of three experiments performed on different blood donors. These data indicate that propionate, but not lactate, inhibits fMLP-induced alkalinization.

noted that there was some donor variability in the effect of propionate on fMLP-stimulated acidification. However, propionate consistently inhibited alkalinization (Table 1 and Fig. 9). As in the F-actin studies, 15-min preincubation conditions had no significant effect on the pH by the end of the preincubation period (relative pH of -0.02 0.05 for lactate and -0.04 0.02 for propionate). Taken together, these results indicate that propionate, but not lactate, inhibits fMLP-elicited cytoplasmic alkalinization. Effect of propionate on N-formyl peptide receptors and G proteins. The previous experiments indicate that propionate, but not lactate, affects fMLP-stimulated cell shape, F-actin localization and content, and cytoplasmic pH oscillations. We therefore investigated some of the possible mechanisms by which propionate could alter these cellular responses. Our initial experiments indicated that propionate-induced cytoplasmic pH and F-actin oscillations were similar to those elicited by fMLP in both magnitude and timing. Given these results, we reasoned that propionate may utilize the fMLP receptor to inhibit fMLP-stimulated responses. We therefore treated cells with the fMLP receptor antagonist tBOC-fMLP. Changes in F-actin content and cytoplasmic pH were measured in cells stimulated with fMLP or propi±

±

onate.

The data in Fig. 10A display the effect of tBOC-fMLP on fMLP- and propionate-stimulated actin responses. Figure

TABLE 1. Effects of SCCA on fMLP-stimulated changes in cytoplasmic pH Maximum change (mean ± SEM)'

Buffer and then 10 nM fMLP (control) 30 mM lactate and then 10 nM fMLP 5 mM propionate and then 10 nM fMLP

Acidification

Alkalinization

% Control alkalinization (mean ± SEM)

-0.04 ± 0.02 -0.06 ± 0.01 -0.04 + 0.02

0.11 ± 0.04 0.14 ± 0.04 0.01 ± 0.02

100 ± 1 8 ± 7b

a In pH units relative to resting pH. b Significantly different from value for cells treated with buffer (control) (P < 0.01).

A

C

T l

2.5

3.0

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z

z

F

2.0

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w

l_

>i 1.5 1

m

1.0

10 M

IMLP

10 MS

fMLP

10 M

fMLP

1.5

1.0

Pro

fMLP

STIMULANT

Propionate

PMA STIMULANT

B

D 0.40

0.4

Buffer

0.35 0.30 0.25

0.3 z

z

0.20 > 0.15 w 0.10 0.05 0.00 -0.05 -0.10 IL

0.2 'a

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0.0

fMLP

~PT _

5Z1

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fUIP STIMULANT

fIMLP

PMA Propionate STIMULANT

FIG. 10. Effect of tBOC-fMLP or PT on SCCA-induced cytoskeletal F-actin and cytoplasmic pH changes. (A) Maximum F-actin induced by fMLP or 5 mM propionate (Pro) in cells incubated with buffer or 10 ,uM tBOC-fMLP (tBOC). In all cases, maximum F-actin content occurred within 10 to 30 s after the addition of fMLP. The value significantly different (P < 0.02) from the value for cells preincubated with buffer on that day is shown with an asterisk. tBOC-fMLP preincubation caused a decrease in baseline F-actin content from 1.0 to 0.54 ± 0.02. In addition, tBOC-fMLP preincubation caused an increase to 0.12 + 0.01 relative pH units above the baseline value. Both of these effects were subtracted from all F-actin and cytoplasmic pH measurements. All results are expressed as the percentages of response in cells preincubated with buffer. Values are means + standard errors of the means of at least four experiments performed on different blood donors. This datum indicates that propionate bypasses the fMLP receptor to induce changes in cytoskeletal F-actin. (B) Maximum changes in pH induced by fMLP or 5 mM propionate (Pro) in cells preincubated with buffer or 10- M tBOC-fMLP (tBOC). In all cases, maximum acidification occurred within 10 to 30 s after the addition of the stimulus, and maximum alkalinization was achieved by 5 min. Values are means + standard errors of the means of at least four experiments performed on different blood donors. The value significantly different (P < 0.02) from the maximum alkalinization in cells preincubated with buffer is shown with an asterisk. These data indicate that propionate bypasses the fMLP receptor to induce decreases in cytoplasmic pH. (C) Maximum F-actin induced by 1 ,uM fMLP, 32 nM PMA, or 5 mM propionate in cells preincubated with buffer or PT. Values are means ± standard errors of the means of at least five experiments performed on different blood donors. The values significantly different (P < 0.02) from the value for cells preincubated with buffer are shown with an asterisk. These data indicate that PT treatment abrogates the propionate-induced F-actin response. (D) Maximum changes in pH induced by 10 nM FMLP, 32 nM PMA, or 5 mM propionate in cells preincubated with buffer or PT. Values are means + standard errors of the means of three experiments performed on different blood donors. The value significantly different (P < 0.02) from the maximum alkalinization achieved in cells preincubated in buffer is shown with an asterisk. These data indicate that PT treatment has no effect on the maximum changes in cytoplasmic pH induced by propionate. 2963

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10A shows that as expected, tBOC-fMLP treatment inhibited F-actin changes elicited by fMLP in a dose-dependent manner. fMLP at 10', 10-8, and 10-6 M fMLP in the presence of tBOC-fMLP exhibited maximal F-actin peaks of 17% ± 4%, 69% ± 6%, and 80% ± 10%, respectively, compared with cells stimulated in the absence of tBOCfMLP. In contrast, tBOC-fMLP treatment had no effect on propionate-induced F-actin. However, it should be noted that tBOC-fMLP alone decreased the F-actin content of unstimulated cells from 1.00 to 0.54 ± 0.02. This had no apparent impact on the propionate- or fMLP-induced response. Taken together, these results indicate that propionate bypasses the fMLP receptor to initiate changes in F-actin. The data in Fig. 10B demonstrate a dose-dependent effect of fMLP in overriding tBOC-fMLP-induced cytoplasmic pH inhibition. The acidification induced by 10-8 and 10-6 M fMLP was inhibited (compared with buffer controls) by 52% ± 18% and 25% ± 20%, respectively. The alkalinization caused by 10-, 10' , and 10 M fMLP was inhibited in the presence of tBOC-fMLP (compared with buffer controls). The percent inhibition values were 96 ± 2, 75 + 8, and 26 ± 6 for 10-9, 10-8, and 10-6 M fMLP, respectively. Propionate induced normal acidification in the presence of tBOC-fMLP (94% ± 3% of propionate alone). Surprisingly, tBOC-fMLP treatment had a significant effect on propionate-induced alkalinization (48% ± 8% of propionate alone). However, tBOC-fMLP alone caused a slow increase in pH (0.12 ± 0.01 units over 5 min). This had no apparent impact on the propionate- or fMLP-induced response. Therefore, propionate bypasses the fMLP receptor to elicit changes in both F-actin content and acidification but may be partially dependent on this receptor to elicit increases in pH. We next considered propionate's interaction with the set of G proteins which have been linked to fMLP-stimulated changes in both F-actin content and cytoplasmic pH (11, 46). Our rationale was that it may be possible for propionate to bypass the fMLP receptor and interact with this set of G proteins to cause inhibition of fMLP-stimulated changes in cytoskeletal F-actin and cytoplasmic pH. We investigated this hypothesis by treating cells with PT, a known inhibitor of the set of G proteins associated with chemoattractant receptors (37). We measured changes in cytoskeletal F-actin and cytoplasmic pH. In these experiments, PMA was used as a positive control. As expected, fMLP-induced changes in F-actin content were abrogated by PT treatment, while those elicited by PMA were left intact (Fig. 10C) (percent maximum F-actin values in PT-treated cells of 17 ± 6 by 10-6 M fMLP and 89 ± 7 by 32 nM PMA). Interestingly, propionate-induced changes in F-actin content were also inhibited by PT treatment (percent maximum F-actin, 6 + 2). These results suggest that propionate-induced changes in F-actin are G protein dependent. Finally, we measured cytoplasmic pH changes in PTtreated cells. Again, as expected, changes in cytoplasmic pH elicited by fMLP were inhibited by PT treatment, while those induced by PMA were not (Fig. 10D) (percent total pH change of 38 ± 8 by 10-8 M fMLP and 90 ± 4 by 32 nM PMA). Unexpectedly, propionate-induced changes in cytoplasmic pH were not significantly inhibited by PT treatment (percent total pH change, 82 ± 6). In fact, the cytoplasmic pH oscillation induced by propionate was not affected by PT treatment in either magnitude or timing (data not shown). These results suggest that propionate can bypass PT-sensitive G proteins to elicit changes in cytoplasmic pH.

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FIG. 11. Hypothetical model for cell activation. Pathways for cell activation are represented by solid lines, whereas hypothetical pathways are indicated by dashed lines. (A) Propionate-induced actin oscillation (transient), localization, and cell shape change. (B) Effect of propionate on fMLP-induced actin localization and cell shape. (C) Propionate-induced decreases and increases in cytoplasmic pH (pHi). (D) Effect of propionate on fMLP-induced increase in cytoplasmic pH. X represents potential intermediaries. Minus signs represent points where propionate may interrupt normal fMLPinduced signal transduction.

DISCUSSION We and others have shown that SCCA inhibit motile functions of PMNs (9, 42-44, 55, 59). Apart from SCCA effects on neutrophils, SCCA can affect functions in other cells. For example, SCCA can inhibit proliferation and histone regulation of cells in culture and can stimulate cytokine production in human monocytes (15, 41, 60). Therefore, SCCA may play an important role in altering normal cell function. This communication investigated both the morphological and the biochemical bases of SCCA effects on neutrophils. In contrast to previous studies (21, 45, 59), SCCA were

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applied to cells in buffered HBSS (pH 7.4), to mimic more closely the in vivo environment. The results indicate that propionate inhibits fMLP-induced actin localization, cell shape, and alkalinization yet has no effect on the F-actin oscillation or acidification. Effect of SCCA on F-actin content, localization, and cell shape. Because cell movement has been linked to F-actin oscillation, the effect of SCCA on F-actin was the subject of our initial investigations (25, 55). It is significant that six SCCA, including propionate, caused both polymerization and depolymerization of actin. In contrast, lactate, an SCCA with a carbon chain length identical to that of propionate, did not produce any alterations in F-actin (Fig. 1 and 2). Propionate and lactate were selected for further study, since they were representative of those SCCA that activate (propionate) and those that did not activate (lactate) actin oscillation (Fig. 1 and 2). We next examined the effects of propionate and lactate on fMLP-induced cell shape, F-actin localization, and content changes. We found that propionate, but not lactate, inhibited the ability of fMLP to cause polarization yet enhanced the immediate increase in F-actin induced by fMLP (Fig. 5 to 8). These results raise at least three possibilities for propionate's inhibition of fMLP-stimulated cell polarization, and actin localization: (i) a direct effect on proteins needed for actin localization and polarization, (ii) a direct effect on signal transduction processes needed for normal actin localization and polarization, or (iii) a nonspecific effect on cytoplasmic pH gradients. The first points will be discussed here, while the last point will be discussed in a later section of this article. Under normal circumstances, the fMLP-induced actin oscillation arises from the assembly of actin monomers. In vitro, this process is controlled by free monomer concentration, [ATP], cytoplasmic pH, [Mg2+], [Ca2+], and temperature (39, 61). Differential association rate constants at either end of the actin filament also regulate filament growth (39). In vivo, a variety of actin-binding proteins help regulate this process by (i) binding to actin monomers to sequester them, (ii) capping filaments to prevent new growth, (iii) stabilizing, protecting, or interconnecting fibers, or (iv) severing actin filaments to provide new polymerization sites (40, 52, 57, 60). In addition, in vitro studies indicate that phospholipids involved in fMLP-initiated signal transduction may regulate actin-binding proteins (29, 52). Our data imply that SCCA do not inhibit actin polymerization but do disrupt the normal localization and thus polarization. Therefore, SCCA must be affecting control mechanisms for subcellular localization of polymerization sites and/or transiently disengaging actin filament capping proteins. Effect of SCCA on cytoplasmic pH. The relationship between cytoplasmic pH and F-actin oscillations is not entirely clear. Our results indicate that a change in cytoplasmic pH alone is not sufficient to cause a change in F-actin. For example, lactate, caproate, and valerate did induce cytoplasmic pH oscillations but did not have any effect on F-actin or cell shape (Fig. 1, 3, and 4). Furthermore, neither propionate nor lactate had any statistical effect on fMLP-induced acidification (Table 1). In contrast, propionate abrogated fMLPstimulated alkalinization (Fig. 9). Finally, lactate had no effect on fMLP-stimulated cell shape or F-actin localization (Fig. 5, 6, 8, and 9). Therefore, these results suggest that SCCA-mediated cytoplasmic pH changes alone are not sufficient to alter fMLP-stimulated changes and support the findings of Grinstein et al. (16), who showed that the

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fMLP-induced F-actin oscillation is unaffected by an internal pH above or below basal levels. However, the importance of fMLP-induced acidification for actin polymerization is controversial. Data from our laboratory demonstrate that when fMLP-induced acidification is equilibrated by the protonophore FCCP, F-actin polymerization is blocked (55). These results imply that fMLP-induced acidification is necessary for fMLP-activated F-actin responses. In contrast, others have found that when fMLP-stimulated acidification is inhibited by the intracellular Ca chelator BAPTA, the increase in F-actin induced by fMLP is unaffected (36). In addition, other studies held the cytoplasmic pH constant in PMNs by (i) suspending intact cells in a high-potassium buffer and using the K ionophore nigericin or (ii) electropermeabilizing cells and loading them with 50 mM HEPES (8). Under both of these conditions, the fMLP-stimulated F-actin oscillation was intact. However, in neither case was the cell shape examined. The differences in the conclusions of the three studies mentioned above may be due to the mechanism used for preventing fMLP-stimulated acidification. For example, it is not clear that a Ca chelator affects cytoplasmic pH oscillation. Similarly, it is not clear how electropermeabilization, high K+ levels, and nigericin affect signal transduction. Our results indicate that fMLP-stimulated acidification is still intact in the presence of propionate, but apparently this is not enough to override the irregular F-actin localization and cell shape changes caused by propionate. In fact, lactate and HCI both cause large degrees of acidification but have no effect on F-actin. Therefore, our data suggest that acidification alone is not sufficient to cause alterations in F-actin mobilization (Fig. 1 to 4). Similarly, the relationship between alkalinization and F-actin polymerization is not clear. The alkalinization induced by propionate or fMLP alone can be attributed to the action of the Na+/H+ antiport located in the plasma membrane (11, 15). Our results indicate that propionate, but not lactate, abrogated fMLP-induced alkalinization (Fig. 9). For example, despite the inhibition of fMLP-stimulated alkalinization in the presence of propionate, an F-actin oscillation can occur. Similar results are obtained using amiloride treatment, which abrogates the increase in cytoplasmic pH but has no effect on F-actin oscillation (33, 55). In contrast, amiloride does inhibit chemotaxis (48). Thus, our data suggest that the processes needed to stimulate cytoplasmic alkalinization may have a role in fMLP-stimulated polarization, since propionate inhibited both alkalinization and polarization. This hypothesis could be tested by treating cells with amiloride and then with fMLP. The data also suggest that propionate-mediated inhibition of chemotaxis may occur through inhibition of alkalinization, as has been shown for amiloride. Finally, these data suggest that propionate's inhibition of the fMLP-stimulated increase in pH and cell polarization must occur before, not during, these events. Effect of tBOC-fMLP on propionate-induced responses. fMLP initiates cellular responses by binding to its receptor. Therefore, a possible target for propionate is the fMLP receptor. We used the fMLP receptor antagonist tBOCfMLP to examine this issue (12). Our results indicate that in the presence of tBOC-fMLP, the fMLP-induced F-actin and cytoplasmic pH oscillations were inhibited. However, the propionate-induced F-actin oscillation was left intact (Fig. 10A). In contrast, tBOC-fMLP had no effect on propionateinduced acidification. However, tBOC-fMLP inhibited propionate-induced alkalinization by 50% (Fig. 10B). The effect of tBOC-fMLP on propionate-induced cell shape and F-actin

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localization still needs to be investigated. Therefore, our results indicate only that propionate can bypass the fMLP receptor to cause actin and cytoplasmic pH oscillations. Another possibility is that propionate affects fMLP receptor binding affinity and receptor number. Some investigators have shown that early responses, such as F-actin oscillation, require smaller amounts of fMLP than later responses such as the oxidative burst (24, 49). In addition, when high concentrations of fMLP bind to their receptors, the receptors convert from low to high affinity and become associated with the cytoskeleton (22, 38). Upon cytoskeletal association, the receptors are thought to be unable to mediate signal transduction. Since propionate inhibits fMLP-induced alkalinization, this suggests that propionate may be causing fMLP receptors to link to the cytoskeleton, therefore requiring more fMLP to cause responses. However, this hypothesis would not explain the fact that fMLP could still activate an F-actin oscillation yet not be able to cause normal F-actin localization. To resolve this paradox, the effect of SCCA on fMLP receptor binding as well as cytoskeletal association must be investigated. This is a testable hypothesis, which our laboratory is currently pursuing. Effect of PT on propionate-induced responses. Another target for propionate's inhibition of fMLP-stimulated responses may be at the level of G proteins. G proteins are linked to fMLP-stimulated changes in both F-actin content and cytoplasmic pH (11, 46). We hypothesized that propionate may bypass the fMLP receptor and interact with G proteins to inhibit fMLP-stimulated changes in cytoskeletal F-actin, cell shape, and cytoplasmic pH. We investigated this hypothesis by treating cells with PT, an inhibitor of the set of G proteins associated with chemoattractant receptors (37). Our results suggest that the propionate-induced F-actin oscillation (Fig. 10C and 11A) and F-actin localization changes (data not shown) were dependent on PT-sensitive G proteins (Fig. 11A). These results imply that propionate may inhibit fMLP-induced F-actin localization by associating with the same set of G proteins involved in fMLP activation

(Fig. liB).

In contrast, the propionate-induced cytoplasmic pH oscillation induced by propionate was not sensitive to PT treatment (Fig. 10D). These results are similar but not identical to those of Naccache et al. (36). Naccache found (as did we) that PT inhibits propionate stimulated F-actin changes. In contrast to the results reported here, Naccache found that PT also inhibited the propionate-induced increase in cytoplasmic pH. We found that the entire cytoplasmic pH oscillation was unaffected by PT (Fig. 11C). The reasons for these differences are unclear but may be explained by differences in experimental methods and buffers. For example, Naccache added unbuffered propionate to the cells, while we added propionate in a pH 7.4 buffer. These results indicate that propionate can elicit cellular responses through PT-sensitive and -insensitive signal transduction pathways. For example, alterations in PMN cytoskeletal actin and calcium can occur independently of PTsensitive G proteins (3, 34). Therefore, the responses elicited by propionate that do not use PT-sensitive G proteins may (i) use another set of G proteins, (ii) use non-G-protein signal transduction pathways such as tyrosine kinases or calmodulin-regulated enzymes (13), or (iii) not require signal transduction mechanisms (Fig. 11D). Other considerations. The present studies examined the effect of propionate on two of the possible signal transduction steps used by fMLP. Another important target for the inhibitory effect of propionate on fMLP-induced responses is

INFECT. IMMUN.

phospholipid metabolism. fMLP activates phospholipase C to hydrolyze phosphotidylinositol bisphosphate, which produces inositol triphosphate and diacylglycerol (26, 37). Inositol triphosphate releases calcium from internal storage sites while diacylglycerol activates protein kinase C (26). Activation of protein kinase C has been linked to activation of the amiloride-sensitive Na+/H+ exchanger which causes increases in cytoplasmic pH (15, 47). Propionate has been shown to cause both an amiloride-sensitive cytoplasmic pH increase and a PT-sensitive calcium oscillation similar to those elicited by fMLP (34). Therefore, propionate also may affect signal transduction components, such as phosphoinositide metabolism, which occurs before activation of the Na+/H+ exchanger. These results, therefore, raise the possibility that SCCA stimulate the release of phospholipid metabolites (e.g., platelet-activating factor, leukotriene B4, etc.) (8). These metabolites could then stimulate the observed actin oscillations. However, three lines of evidence argue against this. First, preliminary results (unpublished observation) indicate that platelet-activating factor receptor antagonists do not inhibit the pH or actin response to SCCA. Second, chemotactic agents stimulate the change in shape to polar (5). However, as shown in Fig. 5 and 6, SCCA evoke change to an irregular cell shape. Finally, if there were a secondary effect, one would expect a delay in the actin response. However, the short-chain-mediated actin and pH oscillations occur in a time frame similar to that of the fMLP-mediated response. Thus, the noted effects of SCCA appear to mediate a direct effect on signal transduction mechanisms. Our results also suggest that SCCA can affect PMN functions needed for host defense. SCCA are known modulators of growth, gene expression, and cell morphology (27, 41). Thus, the presence of SCCA can have a multitude of medical implications, three of which are briefly mentioned here. First, protein turnover is unbalanced in propionic acidemia (30). Second, SCCA, such as butyrate, are associated with colitis caused by certain pathogens in the gut (32). Third, of particular interest to our research group, are the infections associated with periodontal disease. PMNs provide the first line of host defense against these bacterial infections (58). Propionate, butyrate, and other SCCA are found in millimolar concentrations at the sites of periodontal infections (1, 6) and are associated with pathogens thought to be involved with this disease (17, 18, 58). These results suggest that the inhibitory effect of propionate on PMN function may be one mechanism of periodontal disease pathogenesis. Therefore, the effects of SCCA on cellular functions may be important in a variety of pathogenic and disease processes. ACKNOWLEDGMENTS This work was supported by NIH grants DE-08415 and DE-04881. REFERENCES 1. Botta, G. A., C. Eftimiadi, M. Tonetti, T. J. M. van Steenbergen, and J. de Graaff. 1985. Influence of volatile fatty acids on human granulocyte chemotaxis. FEMS Microbiol. Lett. 27:69-72. 2. Brunkhorst, B. A., and R. Niederman. 1991. Ammonium decreases human polymorphonuclear leukocyte cytoskeletal actin. Infect. Immun. 59:1378-1386. 3. Brunkhorst, B. A., G. Strohmeir, K. Lazzari, G. Weil, and E. R. Simons. 1991. Calcium changes in immune complex-stimulated human neutrophils: simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation. J. Biol. Chem. 266:13035-13041. 4. Busa, W. B., and R. Nuccitelli. 1984. Metabolic regulation via

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intracellular pH. Am. J. Physiol. 246:R409-R438. 5. Cassimeris, L., and S. H. Zigmond. 1990. Chemoattractant stimulation of polymorphonuclear leukocyte locomotion. Semin. Cell Biol. 1:125-134. 6. Courtois, P., M. Labbe, M. Pourtois, and I. M. Mandelbaum. 1989. Anaerobes and short-chain fatty acids in crevicular fluid from adults with chronic periodontitis. Bull. Group Int. Rech. Sci. Stomatol. Odontol. 32:19-22. 7. Deim, K., and C. Lentner. 1971. Scientific tables. CIBAGEIGY, Basel, Switzerland. 8. Downey, G. P., and S. Grinstein. 1989. Receptor-mediated actin assembly in electropermeabilized neutrophils: role of intracellular pH. Biochem. Biophys. Res. Commun. 160:18-24. 9. Eftimiadi, C., E. Buzzi, M. Tonetti, P. Buffa, D. Buffa, T. J. M. van Steenbergen, J. de Graaff, and G. A. Botta. 1987. Short chain fatty acids produced by anaerobic bacteria alter the physiological response of human neutrophils to chemotactic peptide. J. Infect. Dis. 14:43-53. 10. Eftimiadi, C., P. Stashenko, M. Tonetti, P. E. Mangiante, R. Massara, S. Zupo, and M. Ferrarini. 1991. Divergent effect of the anaerobic bacteria by-product butyric acid on the immune response: suppression of T-lymphocyte proliferation and stimulation of interleukin-13 production. Oral Microbiol. Immunol. 6:17-23. 11. Faucher, N., and P. H. Naccache. 1987. Relationship between pH, sodium and shape changes in chemotactic factor-stimulated human neutrophils. J. Cell. Physiol. 132:483-491. 12. Freer, R. J., A. R. Dayu, J. A. Radding, E. Schiffmann, S. Aswanikumar, H. J. Showell, and E. L. Becker. 1980. Further studies on the structural requirements for synthetic peptide chemoattractants. Biochemistry 19:2404-2410. 13. Gabig, T., D. English, L. Akard, and M. Schell. 1987. Regulation of neutrophils NADPH oxidase activation in a cell free system by guanine nucleotides and fluoride: evidence for participation of a pertussis toxin and cholera toxin insensitive G protein. J. Biol. Chem. 262:1685-1690. 14. Giloh, H., and J. W. Sedat. 1982. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217:1252-1255. 15. Grinstein, S., and W. Furuya. 1984. Amiloride sensitive Na/H exchanges in human neutrophil: mechanism of activation by chemotactic factors. Biochem. Biophys. Res. Commun. 122: 755-762. 16. Grinstein, S., W. Furuya, and W. D. Biggar. 1986. Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na/H exchange. J. Biol. Chem. 261:512-514. 17. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977. Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg. 18. Holt, S. C., and T. E. Bramanti. 1991. Factors in virulence expression and their role in periodontal disease pathogenesis. Crit. Rev. Oral Biol. Med. 2:177-281. 19. Howard, T. H., and W. H. Meyer. 1984. Chemotactic peptide modulation of actin assembly and locomotion in neutrophils. J. Cell Biol. 98:1265-1271. 20. Howard, T. H., and C. 0. Oresajo. 1985. The kinetics of chemotactic peptide induced changes in F-actin content, F-actin distribution, and the shape of neutrophils. J. Cell Biol. 101: 1078-1085. 21. Iannone, M. A., and G. Wolber. 1989. Effects of adenosine on neutrophil polarization induced by N-formyl-methionyl-leucylphenylalanine, sodium propionate and colchicine. Agents Actions 27:403-406. 22. Jesaitis, A. J., G. M. Bokoch, J. 0. Tolley, and R. A. Allen. 1989. Regulation of chemoattractant receptor interaction with transduction proteins by organizational control in the plasma membrane of human neutrophils. J. Cell Biol. 109:2783-2790. 23. Kalmer, J. R., R. Arnold, M. L. Warbington, and M. K. Gardener. 1988. Superior leukocyte separation with a discontinuous one-step ficoll-hypaque gradient for the isolation of human neutrophils. J. Immunol. Methods 110:275-281. 24. Korchak, H. M., C. Qilkenfeld, A. M. Rich, A. R. Radin, K.

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Propionate induces polymorphonuclear leukocyte activation and inhibits formylmethionyl-leucyl-phenylalanine-stimulated activation.

Short-chain carboxylic acids (SCCA) are metabolic by-products of bacterial pathogens which can alter cytoplasmic pH and inhibit a variety of polymorph...
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