Effect of burning on hepatic phagocytosis as studied in vitro

Pergamoa Press

Life Sciences Vol . 10, Part I, pp. 397-404, 1971 . Printed in Crreat Britain

EFFECT OF BURNING OA HEPATIC PHAGOCYT08IS AS STUDIED IN VITRO B E Schildt and R Houveng Division of Experimental Defence Medicine, Research Institute of Rational Defence, Dept . 1 . S-1Z2 01+ Sundbyberg 4, Sweden

(Received in final form 19 February 1971) A depressed function of the reticuloendothelial systems (RES) ae revealed by impairment of its phagocytic and catabolic functions has recently been reported to suet following various injuries (1) . Possible mechanisms for this depression have been investigated by us and others . Neither an increased corticoeteroid secretion (2) nor s changed blood perfusion rate of the principal RE-organs (3) or liberation of RES depressing substances (4, 5,

6)

or depletion of plasma

factors essential for EES phagocytosis (6, T) could fully explain the results obtained . Interpretation of these

tin vivo

experiments is, however, difficult

since they reflect the "overall-effect" of trauma on the RES . In order to avoid the "multi-factorial" influence exerted upon the RES by trauma in the irtact animal this

in vitro

study was undertaken .

The purpose of the present study was to evaluate the possible effect of burning the liver and plasma donors on

in vitro

hepatic phagocytosis, with special refe-

rence to the possibility of a depletion of essential plus, factors.

Material and Methods CBA male mice were used as liver and plasma donors . Pentotal sodium

(0 .6

x,

60 mg/kg) was given i .p . prior to burning and light ether anesthesia was induced prior to maximal blood letting. Rabbit erythrocytes labelled with S 1Cr(51~._RBC) wex. e employed as test substançe for RES phagocytosis as described elsewhere (8) and suspended in 0 .9 x NaCl to a concentration of 2 g hemoglobin per 100 ml . The inçubation medium contained 2 .25 ml Krebs'-Ringerrphosphate-

397

398

Effect of Burning on Phagocytosis

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bicarbonate (RRPB) (9) and 0 .25 ml fresh heparinized mouse plasma . Liver donor animals were sacrificed by cervical dislocation, the livers removed, washed in pH 7 .4, k °C Krebs'-Ringer solution and approx . 0 .2 - 0 .3 mm thick slices cut

from the median lobe by a Stadie-Rigge microtome (10) . In some donor mice, scald burns were inflicted as previously described (11) sad graded as LD1o " Standard procedure : On the basis of a series of control experiments, the experimental conditions were standardized as follows . To chilled 20 znl beakers wsa added 2 .5 ml incubation medium (HItPB solution + 10 z plasma, pH 7 .5) 50 ul S1Cr~BC suspension (' S x 10~ erythrocytes) and one liver slice (" 125 mg) . The beakers were gassed with 95 x Op and 5 x CO Z , closed and incubated at 37 o C for 20 minutes while oscillating at a rate of 70 cycles/minute . Preliminary tests indicated that a considerable number of S 1 Cr~tBC were adsorbed to the slices without being phagocytized . These cells could conveniently be removed from the slices by hemolysis with saponin . At the end of the incubation period, the liver slices were placed in saponin solution ('Zaponin', Coop er Electronics, 3 drops/10 ml 0 .9 x NaCl) for 10 minutes . They were then washed three times in 25 ~- 0 " 9 x AaCl, transferred to test tubes and the amount of radiochromíum

measured in a well-type scintillation counter . The liver slices were dried over night at 105 oC in a hit-oven, weighed and their specific radioactivity calculated (cimm/mg dry weight) . The figure thus obtained represented the relative phagocytic rate of the liver tissue . Aa the specific radioactivity of our test substance varied from one set of experiments to another, due to the labelling technique used, the experimental values obtained xere always compared with simultaneously performed control experiments . Utilizing the above procedures, experiments were carried out in order to evaluate the effect on tin vitro liver phagocytoais of (cp . Table 1) : a) burning of both liver and plasma donors, b) burning of only liver donors, c) burning of only plasma donors,

d) burning of plasma donors from k hours to 15 days prior

Effect of Burnivg on Phagocytosis

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to collection of plasma

fcp.

399

Table 2) sad e) charigü~g the concentration of plasma

collected from burned or control mice (Table 3) . In all studies, a minimum of six liver slices obtained Prom three different mouse livers were employed in t standard errors were calculated and

each experimental group . Mean values

possible differences compared to simultaneously performed control ezperimeate were evaluated regarding statistical significance by Student's t-Celt .

Results The uptake of S1Cr-BBC by liver slices incubated without plea wen about one half that of slices incubated with 10

x

plasma . The uptake when using livers

and/or plasma from burned mice is summarized in Table 1 . When both liver and plasma originated Prom burned mice, the liver slices contained only about 1/5 (P < 0 .001) . of the amount of test substance found in controls . Liver slices from burned mice incubated with control plasma showed only a slight reduction of the uptake (P > 0.05), whereas control livers incubated with pla®a Prom burned mice showed a significantly (P < 0 .001) reduced uptake compared to controls . These

TABLE 1 Uptake of 51 Cr-BBC by Douse Liver Slices se a Fhnction of Burning the Donors of Livers and/or Plasma .

Number of incubatioas

Treatme~ of Liver donors

Plasma donors

cam/mg Mean ± SE

P2

- (Controls)

- (Co~rols)

3.i t 0 .6

-

6

-

-

2 .2 t o .3

-

8

Blp, ù hrs earlierl

B1o,

0 .5 t 0 .1

< 0.001

6

-

-

5.9 ± 1 .5

-

8

B1o, 4 hrs earlier

-

k .3 ± 0 .~

> 0.05

3.1 t o .1

-

0 .9 t 0 .2

< 0.001

16

6 8

~

-

-

-

Blp,

Blp ~ burn, LD10

4

4

hra earlier

hrs earlier

2) Statistical significance of differences compared to controls .

X00

Effect of Burning on Phagocytoeis

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results demonstrate that the reduced uptake Was due to the poet-burn pla®e. . Plasma was collected from ~+ hours to 15 days post-burn in order to find out how soon after burning normal capacity to promote in vitro liver phagocytoais recovered . Table 2 summarizes the results .

TABLE 2 Influence of a Time Interval between Banning and Collection of Plasma from Burned Mice Added to the Incubation Medium on Uptake of S1 Cr-RHC by Mouse Liver Slices . Time post-~rarn for collection of plasma 4 hours

cpm/mg, Mean ± SE Control (C) 3 .1

± 0 .3

Horned (B)

Ratio B/C

0 .9 ± 0 .2a)

0 .3

4 .5 t 0 .2d) 7 .0 ± o .5d)

1 .0 2 .0

2 days

4 .k ± 0 .5 7 .h ± 0 .6

ù days

2 .2 ± 0 .2

k .3 ± 0 .5b)

7 says

2 .6 f o .5

4 .6 ± 0 .6°)

1 .8

15 daYg

5 .9 ± 0 .3

8 .0 t 0 .7c)

1 .4

2k hours

0 .9

Statistical significance of differences compared to control a : s) P < 0 .001 ; ó) P < 0 .01 ; c) P < 0 .05 ; d) P > 0 .05

The absolute values (cpm/mg) on the uptake of the test substance vary among controls because of differences in specific activity between the daily preparations of 51 Cr~tBC . In order to be able to compare different experimental groups With each other, the ratio between the uptake in the paired groups of burned and controls was calculated (Table 2, Figure 1) . This ratio, being 1 .0 sn controls, was 0 .3 when plasma taken 4 hours post-burn was used indicating a decreased capacity of playa to promote phagocytoais . When pla®a collected 1 - 2 days after scalding was used, the ratio was close to 1 .0 indicating a normal capacity of plasma to promote phagocytoais . Pla~ta collected from mice burned 4 - 15 days earlier augmented the uptake significantly as compared to control plasma. indicating a supra nflrmal concentration of phagocytosis-promoting factors in post-

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401

Eifett ai Horning on Phagocytosis

burn convalescent plasma . In an attempt to find out if the low uptake by slices incubated rrith plasma collected in the early poet-burn period was caused mainly by a shortage of phagocytosis-promoting factors or the presence of RES-depressing agents, the concentration of plasma in the incubation medium was changed from 0

S

to 20

S

(Table 3) "

TABLE 3 Uptake of 51 Cr-RBC by Mouse Liver Slices .as a Function of the Concentration of Plasma from Burned or Control Mice in Incubation Medium .

Conc . of plasma is incubation medium

Plasma collected Pram Burneda) (

0

Controls

B/C

4.8 t o .4 ) 8 .1 t o .7°)

5 .1 ± o .3 b)

5 .1 t 0 .4b)

10 .6

t 1 .40 )

.6 0 .5

s) LD10 scald burn, 2 hrs earlier . Statistical significance compered to : b) corresponding controls P < 0 .01 c) incubation without plasma P < 0 .01

Discussion To judge from the results of the present in vitro ezperimenta, the important role of certain plasma factors for liver phagocytosis is demonstrated . Furthermore, the results indicate that plasma collected during the first day post-burn wen inferior in promoting phagocytosis, while plasma collected 4 - 15 days after burning promoted phagocytosis more effectively than control plasma . Previous reports are thereby confirmed and extended (4,

7,

12, 13) . Ollodart b Manaberger

(4) thought that the decreaséd bacterial resistance seen after hypovolemic shock is man and the dog, wen due in part to a reduced capacity of plasma to promote phagocytosis . Recently Saba (7)

demonstrated similar findings 30 minutes

402

Effect od Horning on Phagocytosis

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FIGURE 1 In vitro hepatic phagocytosis (ratio between cpm/mg in experimental and control groups) plotted together with in vivo phagocytic index (K ~ disappearance rate constant for i .v . injected S 1 Cr-RHC) in mice during 15 days poet-burn (1) .

after abdominal operative procedures in the rat as indicated by the deficient uptake of a test substance by liver slices in plasma . In principal, the low uptake by liver slices incubated together xith plasma collected 2 hours after a burn may be due to either (1) shortage of phagocytosispromoting plasma factors or (2) presence of RES-depressing agents . Our results (Table 3) support the former alternative since the post-burn plasma neither promoted nor depressed the uptake, whereas control plasma enhanced the uptake compared to that obtained by liver slices incubated without plasma . While agreement generally eziste that the plasma factors promoting bacterial phagocytosis are specific immunoglobulins, disagreement prevails regarding the specific nature of plasma factors promoting phagocytoeis of test particles like

Vol . 10, No . 7

Effect oaf Horning on Phagocytosis

40H

gold, carbon, serum albumin aggregates sad lipid emulsion (1ù - 20) . Tn experiments complementary to the present study we subjected plasma to ultrafiltration, barium sulphate adsorption (21) or heat-treatment prior to incubation . The preliminary results indicate that the plasma factors promoting the uptake of 51 CrRBC by liver slices are proteins . If tested in

vitro,

changes in the phagocytic function may go unnoticed or be

erroneously registered if a distinction is not made between adsorption and phagocytosis of the test substance . Although the former invariably precedes the latter, they may reflect entirely different phenomena . Others have tried to avoid this error by trypsin-deo~yribonuclease treatment (22) or vigorous shaking (23) . As erythrocytes were used by us, we were able to utilize the lytìc effect of saponin for the destruction of adsorbed erythrocytes . By careful xashing of the slices thereafter, the contribution to the radioactivity of the slices by diffusable S 1 Cr could be kept at a lox level . After this treatment, the liver slices in control exper~.ments contained approximately 30 ~ of the total amount of radioactivity added to the incubation medium . Tn conclusion , we have shown that the RES-depressing effect of a moderate burn injury, previously reported to occur in mice also

in vitro .

enhanced

in vivo (1),

can be demonstrated

The presence of fresh plasma is the incubation medium greatly

in Vitro

hepatic phagocytosis . On the basis of the results presented,

together with our earlier findings, it seems probable that a shortage of phagocytosis-promoting plasma proteins is the main cause of the decreased phagocytic capacity seen post-burn . This suggestion is supported by the fact that the decrease of the

tin vitro

uptake approximately paralleled that of phagocytosis

in vivo (Figure 1) . Of particular interest in that respect is the finding that the capacity of plasma to promote phagocytosìs was supra-normal 4 and 15 days poet-burn, at which time hyperphagocytoais earlier was found to occur in the intact animal .

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Acknowledgements The skillful technical assistance of Mrs K Wiadh is greatly acknowledged .

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~LQ,

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