Dehydration and factors affecting pulmonary antibacterial defenses

JOURNAL

OF SURQICAL

RESEARCH

16, 44-49

Dehydration

(1974)

and

Factors

Antibacterial JOHN THOMAS

F.

MULLANE, 0.

PHELPS,

M.D., B.S.,

PH.D., M.S.,

Division of Surgery, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. 20012; and Channing and Thorndike Memorial Laboratories, Harvard Medical Unit, Boston City Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts. Presented at the annual meeting of the American Federation For Clinical Research, Atlantic City, April 2%29, 1973. In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care,” as promulgated by the Committee on the Guide for Laboratory Animal Facilit,ies and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences, National Research Council. Address reprint requests to Dr. J. F. Mullane, 685 3rd Ave., New York, N.Y. 10017. Submitted for publication June 5, 1973.

44 @ 1974 hy Academic Press, Inc. of reproduction in any form reserved.

Pulmonary

Defenses

SINCE IMPAIRED INTRAPULMONARY bacterial inactivation of Staphylococcus aureus has been described in rats which were dehydrated for 38 hr [ 141, dehydration could have a direct adverse effect on intrapulmonary host defenses in man. The purpose of this study was to see which of several factors known to influence host responses to bacteria in the lung were altered by dehydration in the rat [3, 4, 10, 11, 171. The effect of 38 hr of dehydration on arterial pH, pCO,, PO,, and osmolality, surfactant, number of viable macrophages recovered by bronchial lavage, in vitro oxygen consumption of the alveolar macrophage, and acid phosphatase, p glucuronidase, and leucine aminopeptidase content of the alveolar macrophage were evaluated.

Copyright .I11 rights

Affecting

ROBERT AND

GARY

G.

WILFONG, L.

HUBER,

B.S., M.D.

METHODS Male Wistar rats weighing 180-310 g were divided into two groups (control and experimental) with similar mean weights. Control rats had free access to food and water. Experimental rats had access to food but not water. All experiments were completed after approximately 38 hr of dehydration. Arterial chemistries, pH, pCO,, and ~0,. Plasma sodium, potassium, osmolality, and creatinine were studied in 20 pairs of control and dehydrated rats that were anesthetized with ether and had blood drawn from the abdominal aorta. Plasma sodium and potassium were measured by a flame photometer (Instrumentation Laboratory), osmolality by an osmometer (Fiske), and creatinine by an autoanalyzer (Technicon). In another group of 24 control and 26 dehydrated rats, arterial pH, pCO,, and ~0, were measured with a pH/pCO,/pOz analyzer (Corning Scientific Instruments). Carotid arterial samples were drawn from conscious rats as previously described [ 14-161. Surfactant. The effect of dehydration on surfactant was measured by changes in alveolar bubble stability ratio [2, 5, 181. The lungs were removed and alveolar bubbles expressed into a hanging drop of aerated water as described by Edmunds and Huber [2]. At least eight bubbles, 30-60 pm in size, from each animal were photographed and 20 min later, were rephotographed. The developed film was projected on a screen

HULLANE

ET

-41~. : DEHYDRATION

so that final magnification before measurement was 2280x. The stability ratio of alveolar bubbles was calculated from the percentage change of the surface area of each bubble. Since surface area of a sphere is equal to sdl, the bubble stability ratio was calculated from the ratio of final to initial bubble diameters squared (&?/d,” X 100). These percentages were averaged for each rat when the stability ratios of 15 control and 21 dehydrated rats were compared. Macrophage studies. For studies of (1 i number and viability of recovered macrophages, (2) macrophage in vitro oxygen consumption, and (3) acid phosphatase, p glucuronidase, and leucine aminopeptidase content of macrophages, free cells of the lung were recovered as previously described from three separate groups of control and dehydrated rats [ 161. However, for enzyme studies, the refrigerated centrifuge was kept at 4°C instead of at 15°C. The number and viability (supra-vital staining with trypan blue) of cells recovered from 13 control and 14 dehydrated rats were determined on a hemacytometer. A smear of recovered cells was made for differential counts. When in vitro oxygen consumption of alveolar macrophages was studied, cells of three rats were pooled and reconstituted with 3 ml of a solution comaining 85% Krebs-Ringer phosphate (pH 7.4) and 15% pooled serum. In vikro oxygen consumption of alveolar macrophages was then measured by a biological oxygen monitor (Yellow Springs Instruments), Total cell counts of each pooled sample was determined and results were expressed as microliters oxygen consumed per hour per million cells (J O,/hr/lO” cells). Nine pooled samples were studied for both control and dehydrated rats. When enzyme content of alveolar macrophages was studied, cells of six rats were pooled and reconstituted with heparinized saline (1.25 units heparin/ml) The pooled sample was recentrifuged at 600 g (4’C) for 10 min and the supernatant removed.

AND

LUNG

HOST

DEFENSE

45

The cells were reconstituted with 4 ml of 0.25 M sucrose and, while surrounded by ice, were homogenized (Virtis-23) for 6 min at medium speed. The homogenized cells were then centrifuged at 12,000 g (4°C) for 15 min. The supernatant was discarded and the homogenate was reconstituted with 3 ml of heparinized saline. The final solution was then frozen and thawed six times bcfore enzyme studies were performed. The enzyme content of the discarded supernatants of the homogenized sample and the original pooled sample were studied in a separate group of 36 control rats (6 pooIed samples) to demonstrate that lysosomal enzymes were not released till the final freeze and thaw process. Sigma Chemical methods were used to determine acid phosphatase (Method 104, revised August 1971), p glucuronidase (Method 105, revised December 1958), and leucine aminopeptidase (Method 251, revised October 1972). Total protein content of each pooled sample was quantitated by a modification of the Kingsbury-Clark urine protein determination. Results were expressed as pmole p-nitrophenol/min/lOO fig protein for acid phosphatase, as pg phenophthalein/min/lOO pg protein for p glucuronidase, and as pmole p-naphthylamine/min/lOO mg protein for leucine aminopeptidase. Five pooled control and six pooled dehydration samples were used to evaluate the effect of dehydration on the enzyme content of macrophages. Presentation of data. All results are expressed as a mean plus or minus the standard error of the mean. Statistica significance was determined by the t test for unpaired data. RESULTS Weight change. Baseline weights for control (245 + 5 g) and dehydrated (237 T 6 g) rats were simiIar. Thirty-eight hours later, dehydrated rats had lost 28.7 T 1.8 g, while control rats had gained 7.7 T 1.3 g (P < .OOl). Arterial chemistries, pH, pCO,, and pOs. Dehydrated rats had a significant increase

46

JOURNAL

OF

SURGICAL

RESEARCH,

Table

1. Arterial

Test Osmolality (mosmjiter) Sodium (mequiv/liter) Potassium (mequiv/liter) Creatinine (mg %)

Table

2. Rubble

Stability

Group 93.8 76.9

Dehydration

299 T 1.7 140 T 0.6 3.7 $ 0.1 0.48 f 0.01

315 ‘i: 148 f 3.3 f 0.54 f

f 2.8 T 6.9 <.05

Cell

Counts,

Total cell count ( X 106) Nonviable cells (%) In vitro 02 consumption (~1 Oz/hr/106 cells)

Table

Solution Homogenized Freeze-thaw

supernatant solution

4.

Acid

1974

P 2.2 0.9 0.1 0.01

<.OOl <.OOl <.OOl <.OOl

DISCUSSION Thirty-eight hours of dehydration adversely affected intrapulmonary mechanisms of antibacterial host defense [14]. Control rats inactivated 91% of a bacterial challenge by 14 hr while dehydrated rats inactivated 78% ; the correlation between fractional lung water content and intrapulmonary bacterial inactivation was significant (P < ,001) [14]. In addition to deViability,

and Oxygen

Control

Test

JANUARY

macrophages was also similar for control and dehydrated rats (Table 3). Macrophage enzyme content. In control studies, acid phosphatase, ,B glucuronidase and leucine aminopeptidase were not released into the pooled sample supernatant and only a trace amount of acid phosphatase was present in the supernatant of the homogenized samples (Table 4). Dehydration had no effect on lysosomal enzyme content. Acid phosphatase, ,L?glucuronidase, and leucine aminopeptidase were similar in macrophages of control and dehydrated rats (Table 5).

Ratio

3. Macrophage

1,

Control

in plasma osmolality, sodium and creatinine and a decrease in plasma potassium (Table 1). Control and dehydrated rats had similar arterial pH (7.36 f 0.01 vs 7.36 + 0.01, P > .8), pC0, (36.0 f 0.9 vs 34.4 f 0.9, P > .2), and ~02 (75.2 T 1.S vs 79.1 T 1.8, P > .l). Bubble stability ratio. Surface properties of 30-60 pm bubbles were affected by dehydration. There was a significant decrease in the bubble stability ratio (Table 2). Macrophage viability and oxygen consumption. When stained smears of cells recovered by bronchial lavage were examined, 95-98s of cells on each slide were monocytes. Cells recovered by lavage were similar in number and viability for control and dehydrated rats (Table 3). In vitro oxygen consumption of pooled samples of recovered Table

NO.

Chemistries

Ratio

Control Dehydration P

16,

VOL.

Consumption

Dehydration

P

5.9 2.3

7 0.7 T 0.2

6.4 2.1

F 0.5 f 0.3

>.5 >.5

1.9

T 0.2

1.7

‘F 0.2

>.4

Phosphatase

of

pmole p-nitrophenol/min/lOO pg protein

pmole p-nitrophenollmin 0.054 8.4

f

Macrophages

0.017

+ 1.1

0.063

f 0.010

MULLANE

ET

Table

AL.:

DEHYDR,4TIOX

5. Enzyme

Content

AND of Pooled

Macroph,age

Control

Enzyme Acid phosphatase (rmole p-nitrophenol/min/lOO p glucuronidase (pg phenophthalein/min/lOO Leucine aminopeptidase (pmole p naphthylamine/min/lOO

LUNG

rg protein) rg protein) mg protein)

creased lung water with dehydration, weight loss, increased plasma osmolality, and abnormal pulmonary surfactant were present and could possibly have affected host defenses. The reduced bactericidal activity with dehydration was not related to systemic hypoxia, acidosis, or to decreased number, viability, oxygen consumption, or enzyme content of recovered macrophages. Since 38 hr of starvation had no effect on intrapulmonary host defenses [14], it is likely that decrease in body water was t.he factor accompanying weight loss that contributed to impaired intrapulmonary bacterial inactivation in the dehydrated rat. Intrapulmonary mechanisms of host defense were also altered after aspiration of blood and this impairment was associated with decreased fractional lung water content [ 161. The increase in plasma osmolality might also affect host defenses since the nephrectomized rat had increased plasma osmolality (297 vs 346 mosm/liter; P < .OOl) and impaired intrapulmonary bacterial inactivation [3]. However, acidosis was present after nephrectomy and ammonium chloride induced acidosis will impair intrapulmonary bacterial inactivation without affecting plasma osmolality (302 vs 302 mosm/liter; P > .9) [3]. The surfactant fraction recovered from the lung by bronchial lavage is needed for normal in vitro bactericidal activity of the alveolar macrophage [ 111. The surfactant fraction will also reverse impaired bactericidal activity of macrophages recovered rats exposed to 100% oxygen for 48 hr [9]. Whether surfactant or some other component of the alveolar lining fluid is needed

HOST

I)ElJEh-SE

47

Samples

lIehydration

P

0.073

f

0.008

0.077

f 0.003

>.6

0.326

f 0.049

0.348

T 0.025

>.6

f 0.22

2.78

7 O.li

>.4

2.56

for bactericidal activity of the alveolar macrophage has not been established. Since surface active forces were abnormal after 38 hr of dehydration, it is possible that abnormal surfactant fraction was contributing to the impaired in vivo bactericidal activity of the alveolar macrophage. Dehydration had no effect on the characteristics of the alveolar macrophage that were studied. The number and viabilit’y of recovered alveolar macrophages were unchanged. Alveolar macrophages have a high aerobic metabolism and are dependent on oxidative metabolism for phagocytosis [17]. However, dehydration had no effect on in vitro oxygen consumption of the alveolar macrophage. In addition, when some of the enzymes that contribute to the actirity of the phagolysosome were studied, the enzyme content of alveolar macrophagcs from control and dehydrated rat,s were similar. These results suggest that the alveolar macrophage was capable of phagocytic and bactericidal activity, but lacked a cofactor, perhaps within the alveolar lining fluid, that was necessary for the normal activity of the macrophage. Although a change in the surface lining material could be responsible, multiple additional variables besides surfactant were not controlled and could be influencing intrapulmonary bacterial inactivation. Complement, cytophilir antibody, and other humoral substances in the alveolar lining fluid could be affected by dehydration. Since subcutaneous epinephrine will impair intrapulmonary bacterial killing by the murine alveolar macrophage [I], it is possible that increased blood concentrations of various hormones

48

JOURNAL

OF

SUHGICAL

RESEARCH,

secondary to dehydration might, also have an effect, on pulmonary host defenses. Although correction of dehydration was not tested in this or the previous study, treatment of fluid deficits would appear to be an important part, of patient management, to prevent bacterial pneumonia in the acutely ill patient. Multiple additional factors may also be present. in the injured or postoperative patient that might impair pulmonary host, defenses. In addition to maintaining adequate hydration, hypoxia acidosis [3], aspiration [16], stress [31, [ 121, hypergIycemia [ 71, hypotension [ 14 ] , renal failure [3], oxygen toxicity IS], and liver damage [8, 131 are to be avoided, prevented, or treated if intrapulmonary host defensesare to be optimal. SUMMARY Since impaired intrapulmonary bacterial inactivation of Staphylococcus aweus has been described in rats which were dehydrated for 38 hr, the effect, of dehydration on several factors known to influence host responses to bacteria in the lung were studied. After 38 hr of dehydration, arterial pH, pCO,, and pop were unchanged, but plasma osmolality was increased. Surfactant, measured by the bubble stability ratio, was impaired by dehydration. When macrophages were recovered from the lung by bronchial lavage, their number, viability, oxygen consumption, and enzyme (acid phosphatase, /3 glucuronidase, leucine aminopeptidase) contents were similar to controls. Since normal oxidative metabolism is needed for phagocytosis and normal enzyme content is needed for lytic activity, the alveolar macrophage appears capable of bactericidal activity. A cofactor, perhaps within the alveolar lining fluid, may be necessary for normal activity of the macrophage and was adversely affected by dehydration. Since in vitro studies have demonstrated the need for normal surfactant fraction for maximal bactericidal activity of the alveolar macro-

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NO.

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1974

phage, the abnormal surface lining material with dehydration may have contributed to the impaired in vivo host defenses. REFERENCES 1 Davis, G. S., Newcombe, D. S., and Green, G. M. Inhibition of bacterial killing by epinephrine and aminophylline in the murine lung: possible role of cyclic-AMP. Clin. Res. 21:659, 1973. 2. Edmunds, L. H., Jr., and Huber, G. L. Pulmonary artery occlusion. I. Volume-pressure relationships and alveolar bubble stability. J. Appl. Physiol. 22:990, 1967. 3. Goldstein, E., Green, G. M., and Seamans, C. The effect of acidosis on pulmonary bacterial function. J. Lab. Clin. Med. 75:912, 1970. 4. Green, G. M., and Kass, E. H. Factors influencing the clearance of bacteria by the lung. J. Clin. Invest. 43:769, 1964. 5. Huber, G. L., Mason, R. J., Boyd, A. E., and Norman, J. C. Experimental pulmonaq hyaline membrane disease following disseminated intravascular coagulation. In G. D. Zuidema and D. B. Skinner (Eds.), Cztrrent Topics in Surgical Research, Vol. 1, p. 411. New York: Academic Press, 1969. 6. Huber, G. L., and La Force, F. M. Progressive impairment of pulmonary antibacterial defense mechanisms ‘associated with prolonged oxygen administration. Ann. Int. Med. 72:208, 1970.

7. Huber, G., O’Connell, D.. Chen, L., Mullane, J., and La Force, M. Experimental diabetes mellitus asd pulmonary antibacterial host defense mechanisms. C/in. Res. 20~530, 1972. 8. Huber, G., O’Connell, D., Libertoff, J., Chen. L., Mullane, J., and La Force, M. Impairment of pulmonary alveolar macrophage function following experimental liver injury. Gastroenterology 62:871, 1972. 9. Huber, G. L. unpublished data. 10. Hurst, D. J., and Coffin, D. L. Ozone effect on lysosomal hydrolases of alveolar macrophages in vitro. Arch. Znf. Med. 127:1059. 1971. 11. La Force, F. M.. Kelly. W. J., and Huber. G. L. Stimulation of bactericidal activity of alveolar macrophages with surfactant fraction. Amer. Rev. Resp. DU. 108:784, 1973. 12. Mullane, J. F., Wilfong, R. G., La Force, F. M., and Huber, G. L. Effect of acute stress on pulmonary host defenses. C/in. lies. 20:580. 1972. 13. Mullane, J. F., Popovic. N. A.. La Force, F. M., and Huber. G. L. Influenre of liver damage on the lung. Chest 62:372, 1972. 14. Mullane, J. F., La Force, F. M., O’Connell,

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ET

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DEHYDliAT~ON

D. M., and Huber, G. L. Acute blood loss and pulmonary host defense mechanisms in the rat. J. Surg. Res. 14:228, 1973. 15. Mullane, J. F., Smith, J. C., and Wilfong, R. G. Hypoxia and stress ulcer formation in the rat. Surgery 74:326, 1973. 16. Mullane, J. F., Popovic, N. A., La Force, F. M., Wilfong, R. G., Bielke, S. R., O’Connell, I>. M., and Huber, G. L. Aspiration of blood

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LUNG

HOST

DEFENSE

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and pulmonary host defense mechanisms. Ann. Surg. 1974, in press. 17. Oren, R., Farnham, A. E., Saito, K., Milofsky, E., and Karnorsky, M. L. Metabolic patterns is three types of phagocytizing cells. J. Cell Biol. 17:487, 1963. 18. Pattle, R. E., and Burgess, F. The lung lining in some pathological conditions. J. Pathol. Bacterial. 82:315,1961.