Vol. 21, No. 2

INFECTION AND IMMUNITY, Aug. 1978, p. 448 457

0019-9567/78/0021-0448$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Distribution of Endotoxin (Lipopolysaccharide) in the Tissues of Lipopolysaccharide-Responsive and -Unresponsive Micet ROBERT A. MUSSON,4 DAVID C. MORRISON,* AND RICHARD J. ULEVITCH Scripps Clinic and Research Foundation, Department of Immunopathology, La Jolla, California 92037

Received for publication 22 February 1978

We examined the distribution of bacterial lipopolysaccharide (LPS) in LPSresponsive (C3H/St) and LPS-unresponsive (C3H/HeJ) mice. The results reported here demonstrate that the rates of removal of an immunological or a toxic dose of LPS from the circulation are the same in both strains of mice. C3H/St spleens accumulated significantly more LPS than C3H/HeJ spleens after the intravenous injection of either an immunogenic or a toxic dose of LPS. There was also a greater amount of LPS associated with cells teased from C3H/St spleens compared to those from C3H/HeJ spleens. After a toxic dose of LPS, there was more LPS in C3H/St lymph nodes, adrenals, lungs, kidneys, and heart than in the corresponding C3H/HeJ tissues. The accumulation of more LPS in tissues from C3H/St mice compared to C3H/HeJ mice suggests that these tissues are involved in the pathophysiological and, ultimately, the toxic effects of LPS. The differential accumulation of LPS in the tissues of these two strains may be the reason for the decreased responses of C3H/HeJ mice to LPS.

Lipopolysaccharide (endotoxin, LPS), in ad- with the appearance of la antigen on their B dition to its pathophysiological and toxic effects cells (45) or Thy 1 antigen on their precursor T (14, 17, 19), elicits a large number of immunolog- cells (15). It has been reported that spleen cells from ical responses in vitro as well as in vivo (16, 28). The 50% lethal dose (LD5o) in mice can be as both C3H/HeJ and responder mice can bind the low as 60 Itg or greater than 2 mg (41), depending same amount of LPS in vitro; and these results on the mouse strain and the LPS employed. suggest that although C3H/HeJ mice are able Doses as low as 0.1 Itg/ml in vitro will induce to bind LPS, they are unable to convert LPSpolyclonal antibody formation (48), and as little cell interactions into a cellular response (13, 37, as 0.1 ,ug will initiate specific antibody formation 48). However, Coutinho et al. (6) detected fewer in vivo (31). At higher concentrations (5 to 100 "LPS-receptors" on C3H/HeJ spleen cells using ,ug/ml), LPS induces mouse spleen cells to max- rabbit antisera against B cells from LPS-responimally synthesize DNA in vitro (37). These re- sive mice (C3H/Tif) that was absorbed with sponses to LPS are under genetic control (26, 33, C3H/HeJ tissues. It is not known if the results seen in these in vitro experiments occur in vivo 47, 48). C3H/HeJ mice have a defect (9, 26, 33, 46-48) or are related to the decreased effect of LPS in which makes them significantly less responsive vivo in C3H/HeJ mice. Other mechanisms could to LPS compared to many syngeneic strains be responsible for the decreased toxic effects and (C3H/St, C3Heb/FeJ, C3H/DiSn, and C3H/ immune response in vivo. For example, a more HeN). C3H/HeJ mice are much less susceptible rapid removal of LPS from the circulation by to the toxic effects of LPS (26, 38, 41), have an C3H/HeJ mice or a shift in the distribution of abnormal inflammatory response to LPS (20,38, LPS in unresponsive mice such that LPS does 39), cannot respond to the adjuvant effect of not reach critical tissues could explain the lack LPS (38), do not mount a polyclonal antibody of an effect in these mice. Therefore, we have response to LPS (7, 48), have a transient specific used LPS, radiolabeled by a new procedure (R. antibody response (which occurs only over a J. Ulevitch, Immunochemistry, in press), to very narrow range of LPS concentrations [35, measure and compare (i) the rate of disappear48]), are unable to respond mitogenically in vitro ance of LPS from the circulation and (ii) the to LPS (7, 36, 42, 47), and do not respond to LPS distribution of LPS in various tissues of C3H/St (responder) and C3H/HeJ mice. The results of t Publication no. 1343 from Scripps Clinic and Research these experiments demonstrate that, whereas no Foundation. * Present address: Department of Pediatrics, National Jew- significant differences were detected between ish Hospital and Research Center, Denver, CO 80206. responder and nonresponder mice in the rates of 44E

VOL. 21, 1978

removal of LPS, significant differences were observed in the accumulation of LPS in several tissues. These data may thus provide information on potential target tissues which may contribute to the deleterious pathophysiological effects after administration of bacterial endo-

DISTRIBUTION OF LPS

449

LPS distribution. Mice were injected intravenously with LPS in 0.2 ml of sterile saline (0.9%). At 1 or 24 h after injection, mice were anesthetized with ether and bled by cutting off the tips of their tails. After the collection of 0.5 to 1.0 ml of blood, the mice were killed by cervical dislocation, and the liver, mesenteric, and cervical lymph nodes, spleen, lungs, brain, toxins. a section of skin, and kidneys free of adrenals were removed. In some animals, adrenals were also examMATERIALS AND METHODS ined. The amount of "I in these tissues and their Mice. C3H/HeJ mice were purchased from Jackson weight were determined. Four mice were examined at Laboratory (Bar Harbor, Me.). C3H/St mice were 1 and 24 h for each dose of LPS. purchased from L. C. Strong Laboratory (Sorrento Correction for blood content of tissue. A porValley, Calif.). Female mice, 6 to 8 weeks old, were tion of the LPS in each tissue was due to LPS in the blood in that tissue. To correct for this, the blood used throughout this study. Bacterial LPS. LPS was extracted from Esche- content of each tissue was calculated by two methods richia coli O111:B4 using the hot phenol-water pro- utilizing bovine serum albumin (BSA) radiolabeled cedure described by Westphal and Jann (50) and pur- with 1311 (Na'311, 12 to 25 Ci/mg, New England Nuclear ified as previously described (24). The material used Corp., Boston, Mass.) by the method of McConahey in this study was the lower-molecular-weight fraction and Dixon (18). In the first method, mice were injected of LPS denoted as peak II on Sepharose 4B chroma- with 500 g of [inI]LPS (specific activity, 0.020OpCi/pug) tography (24). After isolation, this material was radio- followed by an injection of labeled ['31I]BSA 5 min labeled with carrier-free NalnI (not less than 17 before the mice were bled, killed, and dissected at 1 or mCi/pg, Amersham Corp., Arlington Heights, Ill.) 24 h as described above. The concentration of BSA in ([lnI]LPS) as described by Ulevitch (Ulevitch, Im- the blood and amount of BSA in each tissue was used munochemistry, in press). The radiolabeled prepara- to calculate the amount of blood in each tissue. By tions were demonstrated to be indistinguishable from using the measured concentration of LPS in the blood untreated LPS in their physical-chemical properties and the calculated volume of blood in each tissue, we (equilibrium density, sedimentation velocity, molecu- calculated that portion of the LPS due to the blood in lar weight) and biological properties (murine B-cell each tissue and subtracted it from the total LPS in mitogenesis, pyrogenicity, and murine lethality). For the tissue. In the second procedure, mice were injected these studies either 14 or 500 pg of LPS, representing with [131I]BSA, and 5 min later they were anesthetized an immunogenic or toxic dose, respectively, were used. and bled and the blood content of their tissues was A trace amount of [l2`I]LPS was included in each dose calculated by measuring the amount of 131I present. to give a final specific activity of 0.714 pCi per pug or The values obtained for blood volume corrections were independent of the method used and were equivalent 0.020 pCi per pg, respectively. LPS toxicity. Groups of 5 C3H/St mice were in- in the two mouse strains. Therefore, LPS had no effect jected with various doses of unlabeled LPS in 0.2 ml on the amount of blood in the tissues examined. Furof sterile saline (0.9%). Deaths were recorded over the ther, in experiments not reported here, we have been next 3 days. At the end of 3 days, the cumulative unable to detect decreases in aortic blood pressures toxicity at each dose was determined. The LD50 was during a 5-h period after injection of an LD5o of this calculated by the method described by Reed and LPS in C3H/St mice. Therefore, hemodynamic Muench (27). In addition, four C3H/HeJ mice were changes induced by the LPS would most likely not injected with 2,000 pg of LPS, and deaths were re- contribute significantly to differences in tissue blood corded for the next 3 days. None of the C3H/St mice content. Normalization of LPS content. The amount of injected with a dose equivalent to or less than the LPS in each tissue is presented either as the percent LD50 died within 24 h after injection. Disappearance of LPS from circulation. Mice of the amount of LPS injected or a normalized value were injected intravenously with LPS in 0.2 ml of R. R was calculated using the following formula: R = saline. Different mice were then bled (0.2 to 0.3 ml) at milligrams of LPS in tissue per gram of 2, 4, 15, and 30 min and at 1, 2, 4, 8, 15, and 24 h after tissue/milligrams of LPS injected per gram of total injection by cutting off the tips of their tails. Two mice body weight. This calculation was previously utilized were bled at each time point, and values obtained by other investigators (2). These normalized values usually agreed to within 10%, except at the very late were used to compare LPS accumulation in the two time points with the lower LPS dose. The fact that strains of mice since they take into account mouse-toless than 1% of the injected LPS was found to be mouse variation in tissue and body weight. Data are localized at the site of injection after 5 min would presented as the mean ± standard error of R for four argue strongly for total systemic administration. The mice at a given dose and time after injection. The blood was weighed, and the amount of LPS (ug) per mean R values for a given C3H/St or C3H/HeJ tissue gram was calculated based on the "I measured in a were compared by Student's t test. Association of LPS with spleen cells. Twentywell-type counter (Baird-Atomic model 530). The logo of micrograms of LPS per gram of blood was plotted four hours after injection of 14 pg of LPS into six versus time, and straight lines were fit to the data animals of each strain, spleens were removed and using least-square linear regression analysis. Slopes of weighed and the amount of LPS present was determined as before. Cells were then teased ree from the the lines were compared using a t test.

MUSSON, MORRISON, AND ULEVITCH

INFETIBMMUN.

spleens in 4 ml of ice-cold Hanks balanced salt solution supplemented with L-glutanine. The cells were washed twice in 10 ml of the same media. Then, erythrocytes were lysed using a solution of ammonium chloride (29). After a final wash in Hanks balanced salt solution, the amount of LPS associated with the cell pellet was determined. The cells were resuspended and counted in a hemocytometer, and the amount of LPS per 107 cells was calculated.

rapid phase seen with these mice was also complete in about 30 min, and both phases were linear, indicating first order kinetics. The halflife during the later phase for C3H/HeJ mice was 9.55 h. The small difference in first order rate constants (0.065 h-' for C3H/St and 0.073 h-' for C3H/HeJ) is not statistically significant. Disappearance of a toxic dose from the circulation. Figures 2A and 2B demonstrate the kinetics of removal of 500 ,ug of LPS (LD50 in C3H/St mice) from the circulation of C3H/St and C3H/HeJ mice, respectively. The pattern of disappearance was the same as it was for 14 ug of LPS, i.e., an initial rate followed by a slower rate of decrease in the level of LPS are seen. As for the lower dose of LPS, the kinetics of these two phases were first order. The apparent difference in the first order rate constant for the secondary phase of the curves (0.070 h-' for C3H/St and 0.077 h-' for C3H/HeJ) is not statistically different. The rate constants for the secondary decrease in the LPS were independent of the dose of LPS, although the absolute concentration of LPS in the circulation at a given time after injection was greater with 500 1tg of LPS. The mean tl/2 for all the clearance data at both doses from both strains ± standard error (SE) was 9.79 ± 0.35. This 3.6% variation is not significant. Distribution of an immunogenic dose of LPS. The clearance data demonstrated that there is no significant difference in the kinetics

450

RESULTS Since mice respond differently to high and low doses of LPS, we examined the disappearance of both immunogenic and toxic doses in both strains of mice. The toxic dose of the LPS used in these experiments was found to be 470 jg in C3H/St mice and much greater than 2 mg in C3H/HeJ mice. The LPS used in these studies was previously shown to be immunogenic in A/J mice at a dose of 10 ,ug (4). Disappearance of an immunogenic dose of LPS from the circulation. The data in Fig. 1A demonstrate the kinetics of disappearance of 14 ug of LPS in C3H/St mice. After an initial period of rapid clearance, the rate of disappearance from the circulation decreased. The initial phase lasted about 30 min, and the linearity of both phases of disappearance in this plot indicates that the removal was first order. The halflife for the second phase was 10.66 h. In C3H/HeJ mice, removal of the same dose of LPS was also biphasic (Fig. 1B). The initial

1.2-

A.) C3H/St

mW 0.8-

.A 0.44-

0

_.

___

t,=10.6S Heirs _ .=0.0

- 0-

.0.2-

a

.- 1.21.1 C3N/N.J

-a 0.3-

ma

a

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t%=9.55

a

~m

u-

Nomrs

l

0 0

6

4

i 1A 1e (hours)

20

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Time After Ilijectin

FIG. 1. Kinetics of disappearance of a 14-pg dose of LPS. (A) C3H/St mice; (B) C3H/HeJ mice. The correlation coefficient (r) and tl/2 are shown for the secondary phase.

VOL. 21, 1978

DISTRIBUTION OF LPS

of removal of LPS from the circulation. Therefore, the distribution of LPS in these two strains of mice after injection of an immunogenic or toxic dose was examined. As was described in Materials and Methods, the amount of LPS in the blood in each tissue is subtracted from the total amount of LPS in each tissue. Therefore, the R value reflects only the LPS which has adhered to the tissue. Table 1 lists the tissues examined for LPS content after the injection of 14 jig of LPS. After blood cells were removed by centrifugation, greater than 90% of the LPS in the blood at 1 or

451

24 h was in the serum. After 24 h, the level of LPS in the circulation decreased and, in general, the amount of LPS increased in the other tissues was examined. Of the tissues listed in Table 1, the only statistically significant differences in LPS levels (R) between the two strains of mice were in the spleen (C3H/St > C3H/HeJ, P < 0.05) and skin (C3H/HeJ > C3H/St, P < 0.05) at 24 h. After correction for the blood content of each tissue, negative amounts of LPS were calculated for some of the tissues examined. This is attributed to the low levels of LPS in these tissues and

A.1 C3N/St

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tY9.95NHours r=0.97

0

._

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1.8-

._

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1.4-

Ca

,,

i

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I.1 C3N/Hle

2.6-

-0-

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i 12 16 Time after Injectien Ihoursl

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24

FIG. 2. Kinetics of disappearance of a 5X-pg dose of LPS. (A) C3H/St mice; (B) C3H/HeJ mice. The correlation coefficient (r) and the tI12 are shown for the secondary phase. TABLE 1. Distribution of LPS in C3H/St and C3H/HeJ mice after an immunogenic dose of LPSa C3H/St 24h

lhb

Tissue

% In-

jIn-d

C3H/HeJ

~% In-

Rc (mean SE) jecIndc ±

Rc (mean ± SE)

1h % In-

jected" Rc (mean SE)

0.29 2.15 ± 0.47 0.06 0.54 ± 0.33 0.79 ± 0.33 0.67 1.22 0.28 1.49 2.02 ± 0.68 3.43 ± 0.34 0.44 0.91 ± 0.11 0.56 ± 0.55 (0.20) (0.26) ± 0.14 0.03 0.02 ± 0.01 Brain (0.07) (0.03) ± 0.03 (0.08) (0.06) ± 0.02 0.01 ±0.01 0.03 0.19 0.16 ± 0.03 0.07 0.01 0.03 Skin 23.8 5.78 ± 0.46 6.67 2.06 i 0.81 1.57 ± 0.20 9.46 Liver 0.22 i 0.38 0.50 0.74 ± 0.11 0.20 (0.08) (0.3) ± 0.21 Kidney 0.12 0.57 ± 0.08 (0.06) (0.17) ± 0.22 Heart (0.34) (1.27) ± 0.29 1.29 ± 0.37 34.88 4.38 8.71 ± 0.78 6.92 ± 4.38 32.6 Blood a Dose of LPS was 14 yg. Negative numbers are in parentheses; R values in boldface for a different (P < 0.05), C3H/St tissue versus C3H/HeJ tissue. Time after injection. ' LPS content corrected for blood content. d Total blood content in mouse using 6% of body weight as the total blood volume.

Lymph node Spleen Lung

0.15 0.45 2.91

24h % In-

ejected Rc (mean ± SE) 1.12 1.95 ± 0.71 1.10 2.12 ± 0.44 0.79 1.27 ± 0.22 0.09 0.05 ± 0.04 0.47 0.27 ± 0.03 24.54 5.45 ± 0.17 0.40 0.50 ± 0.14 0.19 0.70 ± 0.19 7.96 1.48 ± 0.16 given tissue are significantly

452

INFECT. IMMUN.

MUSSON, MORRISON, AND ULEVITCH

variations in the blood content of a given tissue. adrenals. The normalized R value is dependent Except for C3H/St heart at 1 h, the negative R on the weight of the tissue, and for organs with values were not statistically significantly differ- low weights, accurate weighing is critical. As ent from zero. adrenals are very small and difficult to excise Thirty-one to fifty-three percent of the in- free from all fat tissue, a small error in removing jected material was accounted for by adding up or weighing the adrenals could lead to fairly the radioactivity recovered in all of the tissues large errors in R. For these reasons, adrenals examined. This range of recovery is comparable from six additional mice of each strain were to that seen after the injection of LPS into examined 24 h after a toxic dose injection. Great rabbits (2). care was taken to remove all of the surrounding Distribution of a toxic dose of LPS. The fat tissue. The R values obtained in this experidistribution of a toxic dose (500 ,g) of LPS in ment (26.54 ± 3.47 for C3H/St and 14.34 ± 1.85 C3H/St and C3H/HeJ mice is shown in Table for C3H/HeJ) confirmed the approximate two2 and Fig. 3. As with 14 jag of LPS, most of the fold difference in R values in the adrenals seen LPS was still in the blood after 1 h in both in Fig. 3. The larger R values may be due to strains of mice, and at 24 h the amount of LPS greater care in dissection and thus lower tissue was lower in the blood and higher in the other weights. tissues compared to the levels at 1 h. As with Accumulation of LPS in spleen cells. The the lower dose of LPS, greater than 90% of the above results indicate that more LPS was accuLPS in the circulation was in the serum. Of the mulated in C3H/St spleens than in the tissues listed in Table 2, there was also signifi- C3H/HeJ spleens. It is possible that this differcantly more LPS in C3H/St than C3H/HeJ ence was due to a difference in the amount of adrenals at 24 h (P < 0.01), lymph nodes at 24 LPS associated with the lymphocytes and monoh (P < 0.05), spleen at 1 and 24 h (P < 0.05 and nuclear cells in the spleen. Therefore, we examP < 0.01), lungs at 24 h (P < 0.01), heart at 24 ined the amount of LPS associated with cells h (P < 0.05), and kidneys at 24 h (P < 0.01). teased from spleens from six mice from each These differences are more clearly demonstrated strain 24 h after an immunogenic dose of LPS. by the bar graph shown in Fig. 3. Thirty-one to From 19.7 to 34.4% (26.25 + 2.47%, mean ± SE) fifty-two percent of the injected LPS was ac- of the LPS originally associated with the counted for. Thus, there does not appear to be C3H/St spleens was in the washed cell pellet, any difference between strains in the amount of and 21.7 to 36.2% (27.42 ± 2.54%, mean ± SE) LPS recovered at either dose. Negative amounts was in the C3H/HeJ cell pellets. The results of LPS were also seen at this dose of LPS at 1 h, indicate that there is a 2.6-fold difference bebut none were statistically significant from zero. tween these two strains in the amount of LPS The data in Table 2 and Fig. 3 indicate that per 107 cells (4.45 ± 0.73 ng for C3H/St mice and the amount of LPS accumulated in C3H/St 1.80 ± 0.22 ng for C3H/HeJ mice, mean ± SE) adrenals was greater than the levels in C3H/HeJ which is statistically highly significant, P < 0.01. TABLE 2. Distribution of LPS in C3H/St and C3H/HeJ mice after a toxic dose of LPS' C3H/St Tissue

1hb In~~% InR' (mean ± SE) j.cted' jIend%

C3H/HeJ 24h R' (mean ± SE)

lh % In-

± jected R' (mean SE) je

24h

In-d n

R' (mean ± SE)

Lymph node 0.11 0.49 ± 0.14 0.78 3.33 ± 0.47 0.08 0.26 ± 0.15 0.32 1.54 ± 0.39 Spleen 0.33 0.93 ± 0.18 1.25 2.74 ± 0.29 0.22 0.40 ± 0.03 0.81 1.52 ± 0.13 Lung 0.55 0.48 ± 0.14 0.69 1.17 ± 0.16 (0.02) (0.02) ± 0.29 0.24 0.55 ± 0.13 Brain (0.02) (0.02) ± 0.05 0.09 0.05 ± 0.01 (0.1) (0.05) ± 0.02 0.01 0.01 ± 0.01 Skin 0.14 0.09 ± 0.06 0.58 0.27 ± 0.06 0.24 0.13 ± 0.09 0.49 0.33 ± 0.03 Liver 5.67 1.46 ± 0.47 25.71 5.02 ± 0.59 5.40 1.17 + 0.15 23.40 4.94 ± 0.26 Kidney 0.57 0.29 ± 0.08 0.69 0.74 ± 0.11 (0.4) (0.55) ± 0.15 0.27 0.28 ± 0.04 ± Heart 0.41 1.29 0.45 0.80 0.76 ± 0.13 0.02 0.40 ± 0.22 0.01 0.37 ± 0.02 Blood 43.45 8.27 ± 0.48 8.79 1.54 ± 0.21 46.7 8.71 ± 0.78 6.30 1.22 ± 0.24 Adrenals 0.06 1.85 ± 1.08 ± 0.15 6.89 0.84 (0.01) (0.22) ± 0.12 0.07 3.48 + 0.13 Dose of LPS was 500 ig. Negative numbers are in parentheses; R values in boldface are significantly different for a given tissue and time; spleen at 1 h, lymph node, and heart (P < 0.05); all others (P < 0.01), C3H/St tissue versus C3H/HeJ tissue. 'Time after infection. LPS content corrected for blood content. d Total blood content in mouse using 6% of body weight as the total blood volume. '

VOL. 21, 1978

DISTRIBUTION OF LPS

A

B 10 Blood

C 0 Airemals Lymph Noeis

1Liver

E

F

G

H

ISpleen

Lungs

iHeart

IKidneys

24 24 24 24 Time After Injection (hours)

24

453

8-

6- i AJ -tj

2-

1.

I

I'

1 24

1 24

I1

24

FIG. 3. LPS in C3H/St and C3H/HeJ tissues where there are significant differences in the level of LPS. Shaded bars, C3H/St; open bars, C3H/HeJ. Lines represent the standard error of the mean. R = (LPS in

tissue/gram of tissue)/(LPS injected/mouse weight).

DISCUSSION In this study, we followed the fate of LPS in LPS responsive (C3H/St) and LPS unresponsive mice (C3H/HeJ) by observing (i) clearance of LPS from the circulation and (ii) the distribution of LPS in various tissues. C3H/St and C3H/HeJ mice removed LPS from the circulation at the same rates with similar biphasic kinetics (Fig. 1 and 2). However, there was a significant difference between the two strains in the levels of LPS in the spleen (Table 1) and spleen cells after an imungenic dose. In addition, LPS levels were greater in C3H/St lymph nodes, adrenals, lungs, kidneys, heart, and spleen 24 h after the injection of a toxic dose of LPS (Table 1, Fig. 3). Although in vivo analysis is more complex, it has provided information that can not be obtained in vitro. The mechanism or specific defect responsible for the lack of a response of 03H/HeJ mice to LPS has yet to be determined. A number of mechanisms have been postulated (9,37,44,48), and some of these are incorporated in the schematic daammed below: B)

LEARANCE

(A) LPS-----PROCESSIN -CELL OR TISSUE -.-eRESPONSE (C)

"~Nh)NOWRESPONSE

(A) LPS may be processed differently in 03H/HeJ mice such that the LPS is converted into an inactive form or that C3H/HeJ mice lack a processing function which converts LPS

into an active form. (B) LPS may be cleared more rapidly in C3H/HeJ mice and thus prevent

LPS from reaching critical responsive tissues. (C) Tissues which are targets for LPS may, in C3H/HeJ mice, have a decreased number of receptorsr" necessary for interaction with LPS or may have a receptor of lower affinity such that the amount of LPS required to stimulate a cell does not accumulate on the cell. (D) Target tissues or cells in C3H/HeJ mice may bind LPS, but for one or more reasons this binding is not converted into a response. The siiaiyin the blood clearance data in the two strains suggests that mechanism B is improbable. However, the decreased amount of LPS seen in many 03H/HeJ tissues suggests that mechanism C may be important in explaining the decreased in vivo response of C3H/HeJ mice in LPS. The relationship of other data to the data presented here and to these possible mechanisms is discussed below. Disappearance of LPS from the circulation. The loss of LPS from the circulation followed a biphasic time course. It is important to note that (i) both strains of mice removed LPS from the circulation with the same biphasic ki-

454

MUSSON, MORRISON, AND ULEVITCH

INFECT. IMMUN.

netics and the same rate constants; and (ii) the rate constants for the secondary phase of disappearance were the same for an immunogenic (14 ,Ag) or a toxic (500 ,Lg) dose of LPS. This latter point is especially significant, since it suggests that whatever the mechanism for the secondary phase removal of LPS may be, it is not dependent on the concentration of LPS. Instead, the rate of removal remains the same even when toxic levels of LPS are present. In addition, since the toxic dose of LPS did not decrease the rate of removal, the cause of death is not likely to be a simple poisoning of this clearance system. Even so, it is important to keep in mind that at the toxic dose there was a much higher level of LPS persisting in the circulation. This high level may be the driving force for accumulation of LPS in tissues that subsequently leads to pathological effects and death. In these studies, we did not select for C3H/St mice that were most resistant to the toxic effects of LPS since tissues were examined before any animals died. The biphasic shape of the curves in Fig. 1 and 2 may be explained by a number of possible mechanisms, several of which are considered here. For example the initial rapid decrease may be due to rapid binding of LPS to high affinity sites available to the circulation. The slower rate of disappearance may represent association of LPS with lower affinity sites. High and low affinity sites may simply be different cell populations in contact with the circulation. Other investigators have shown that the cells of the reticuloendothelial system as well as parenchymal cells in the liver are important for the clearance of LPS (11, 51). Alternatively, the biphasic nature of these curves may represent removal of two species of LPS molecules. Several investigators have shown that LPS is physically altered by serum (21,32). Recently, using the same LPS preparations used here, Ulevitch and Johnston (unpublished data) have demonstrated buoyant density changes in LPS after an interaction with rabbit sera either in vivo or in vitro, which result in the loss of some biological activity. This change is essentially complete in 30 min. In preliminary experiments, equivalent shifts in the density of LPS were seen in serum taken from both strains of mice at 1 and 24 h (R. A. Musson, unpublished data). Since the appearance of LPS of a lower density corresponds with a slower rate of disappearance, it may be that native LPS is cleared at the faster rate and that altered LPS is cleared at the slower rate. Inasmuch as this type of processing is the same in both strains, it is apparently unrelated to the reported ability of LPS-responsive mice and the inability of C3H/HeJ mice to convert LPS from an inactive

to an active form (44). Other studies have shown that LPS was initially cleared more rapidly in animals made tolerant to LPS than in nontolerant ones (1, 10). In addition, the level of LPS in the circulation after this initial decrease was lower in tolerant animals (1), and the lower level persisted for a longer period of time in tolerant animals (3). C3H/HeJ mice did not demonstrate this characteristic increased rate of clearance compared to responsive C3H/St mice, and the suggestion would be that the lack of a response by C3H/HeJ mice is not due to a tolerant state. Studies in other species and with LPS from other types of bacteria (1) have also shown the clearance of LPS to be biphasic. The initial rates in these other studies were much more rapid and seemed to mainly be a measurement of reticuloendothelial system activity (11). This may reflect differences in the animal species, type of LPS, or perhaps, most importantly, the amount of particulate material in the LPS preparation. In this regard, it is worth noting the nonparticulate nature of the LPS used here (24). Significantly less LPS was associated with washed cells teased from C3H/HeJ spleens in comparison to those from C3H/St. These data demonstrate that some of the LPS in the spleen has, in fact, strongly associated with cells which may be involved in the immune response and is not simply due to LPS trapped within the connective tissue structure of the spleen. However, these results vary from data obtained in spleen cell binding experiments in vitro, where no differences between C3H/St and C3H/HeJ cells were seen (13, 48). This variance in results suggests that in vitro and in vivo binding studies may measure different types of LPS-cellular association; and/or that factors which are present in vivo, but not in vitro, modulate this interaction. The LD50 for the LPS used in these distribution and clearance studies was 470 jig for C3H/St mice and much greater than 2 mg for C3H/HeJ mice. The distribution of LPS after the injection of the LD50 for responder mice revealed several similarities, as well as differences, between the two strains in the amount ofLPS that is localized in a given tissue. The livers taken from C3H/St and C3H/HeJ mice had the same level of LPS either at 1 or 24 h after injection (Table 2). The liver has been cited as a major organ for the removal (2) and detoxification (25) of LPS. Therefore, the lack of a difference in the levels of LPS in the liver suggests that C3H/HeJ mice are not removing LPS from the circulation at an abnormal rate. The data in Table 2 and Fig. 3 also implicate

VOL. 21, 1978

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several tissues as potential sites for LPS-induced injury or suggest a role for these tissues in the effects of LPS. Greater accumulation of LPS in C3H/St spleen and lymph nodes correlates with the role these tissues play in the immune response to LPS, as mentioned above. Furthermore, the higher levels of LPS in C3H/St spleen and lymph node correlates with the recently suggested role for spleen cells in mediating the toxic effects of LPS (8). This accumulation may also reflect RES activity in these tissues. However, the difference between these tissues in the two strains is unlikely to be due to differences in RES activity in light of the comparable accumulation seen in the livers. The difference seems more likely to be due to the ability of lymphoid elements of these tissues to bind LPS since lymphoid cells are concentrated in spleen and lymph node and not in liver. Adrenals isolated from C3H/St mice 24 h after LPS injection contained twice as much LPS as C3H/HeJ adrenals (Table 2; Fig. 3). It is known that adrenalectomy makes mice up to 6,000-fold more sensitive to the toxic effects of LPS and that cortisone protects against LPS-induced death (5). Thus, increased LPS binding in C3H/St adrenals may lead to a loss in adrenal function. The resulting loss of adrenal hormones and their regulation of various necessary bodily functions may be the cause of death in these mice. Likewise, the lack of LPS binding and therefore normal function of adrenals in C3H/HeJ mice may be one reason these mice survive. The twofold difference in the LPS levels in adrenals was confirmed by eminig adrenals in six additional mice of each strain. After a toxic dose of LPS (Table 2; Fig. 3), there was also significantly more LPS in C3H/St kidneys (2.6-fold), lungs (2.1-fold), and heart (2.0-fold). Accumulation of LPS in the kidneys may

be due to filtration collection of LPS com-

plexes that have formed in the circulation or may represent binding of LPS to kidney tissue. Certainly, LPS-induced Shwartzman reaction involves damage to kidneys (43), and the accumulation seen here may be involved in the priming for or initial damage of kidneys. A previous report demonstrates that LPS can interact in perused kidney to prepare for generalized Shwartzman (12). In addition, significantly greater accumulation of LPS in tissues such

as

lungs and heart of responder mice, which are known to be damaged by injection of endotoxin (43), suggests that concentration of LPS in these tissues may be important in initiating the subsequent necrosis. Localized activation of humoral mediator systems and the consequent accumulation of neutrophils, platelets, and mac-

455

rophages can be initiated by LPS (22, 23). Where there was a significantly greater accumulation of LPS in C3H/St tissues, this difference was consistently 2- to 2.5-fold. Other investigators have shown that a variety of C3H/HeJ cells (macrophages, fibroblasts, T-cells, and others) fail to respond to or have a reduced response to LPS in vitro (15, 30, 34; Watson et al., J. lInmunol., in press). In addition, several of the aberrant responses of C3H/HeJ mice to LPS in vivo have been linked to the product of a single genetic locus (46). Therefore, a defect common to all C3H/HeJ cells which decreases the response to LPS seems likely. A simple interpretation is that there is a basic structural difference common to all C3H/HeJ tissues that is involved in LPS-binding. However, no study to date makes it possible to eliminate mechanism D (see text schematic above) as a possible explanation for the decreased response of C3H/HeJ tissues to LPS. Additional in vitro, as well as in vivo, experiments are required to exmine this point. ACKNOWLEDGMENTS We thank Vicky Byers for her expert technical stance. This work was supported by Public Health Service grants AI 13187 from the National Institute of Allergy and Infectious Diseases and HL 16411 from the National Heart, Lung, and Blood Institute. Robert A. Musson is the recipient of Public Health Service Postdoctoral Research Fellowship AI 0549601, and David C. Morrison is the recipient of Research Career Development Award AI 00081-02, both from the National Institute of Allergy and Infectious Diseases. Richard J. Ulevitch is the recipient of Public Health Service Young Investigator Pulmonary Research Grant HLI 18376 from the Heart, Lung, and Blood Institute.

LITERATURE CTD 1. Braude, A. L 1964. Absorption, distribution, and elimination of endotoxins and their derivatives, p. 98-109. In M. Landy and W. Braun (ed.), Bacterial endotoxins. Rutgers University Press, New Brunswick, N.J. 2. Braude, A. I., F. K. Carey, and M. Zalesky. 1955. Studies with radioactive endotoxin. I. Correlation of physiologic effects with distribution of radioactivity in rabbits injected with lethal doses of E. coli endotoxin labeled with radioactive sodium chromate. J. Clin. Invest. 34:8584866. 3. Carey, F. J., A. L. Braude, and Me Zalesky. 1958. Studies with radioactive endotoxin. Iml. The effect of tolerance on the distribution of radioactivity after intravenous injection of Eschenchia coli endotoxin labeled with CrT5. J. Clin. Invest. 37:441-457. 4. Chiller, J. ML, B. J. Skidmore, D. C. Morrison, and W. 0. Weigle. 1973. Relationship of the structure of bacterial lipopolysaccharides to its function in mitogenesis and adjuvanticity. Proc. Natl. Acad. Sci. U.S.A. 70:2129-2133. 5. Chedid, L., K. Parant, F. Boyer, and R. C. Slarnes. 1954. Nonspecific host responses in tolerance to the lethal effect of endotoxin, p. 500. In M. Landy and W. Braun (ed.), Bacterial endotoxins. Rutgers University Press, New Brunswick, N.J. 6. Coutinho, A., L. Forni, F. Melchers, and T. Watan-

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abe. 1977. Genetic defect in responsiveness to the Bcell mitogen lipopolysaccharide. Eur. J. Immunol. 7:325-328. 7. Coutinho, A., E. Gronowiez, and B. M. Sultzer. 1975. Genetic control of B-cell responses. I. Selective unresponsiveness to lipopolysaccharide. Scand. J. Immunol. 4:139-143. 8. Glode, L M., S. E. Mergenhagen, and D. L Rosenstreich. 1976. Significant contribution of spleen cells in mediating the lethal effects of endotoxin in vivo. Infect. Immun. 14:626-630. 9. Glode, L M., I. Scher, B. Osborne, and D. Rosenstreich. 1976. Cellular mechanism of endotoxin unresponsiveness of C3H/HeJ mice. J. Immunol. 116:454-461. 10. Herring, W. B., J. C. Herion, R. I. Walker, and J. G. Palmer. 1963. Distribution and clearance of circulating endotoxin. J. Clin. Invest. 42:79-87. 11. Howard, J. G., D. Rowley, and A. C. Wardlaw. 1958. Investigations on the mechanism of stimulation of nonspecific immunity by bacterial lipopolysaccharides. Immunology 1:181-203. 12. Husberg, B., F. Linell, T. Nilsson, and H. Fritz. 1975. Renal cortical necrosis induced in rabbits by local perfusion followed by systemic administration of endotoxin. Eur. Surg. Res. 7:230-241. 13. Kabir, S., and D. L Rosenstreich. 1977. Binding of bacterial endotoxin to murine spleen lymphocytes. Infect. Immun. 15:156-164. 14. Kass, E. H., and S. M. Wolf. 1973. Bacterial lipopolysaccharides. University of Chicago Press, Chicago, Ill. 15. Koenig, S., M. K. Hoffman, and L Thomas. 1977. Induction of phenotypic lymphocyte differentiation of LPS unresponsive mice by an LPS-induced serum factor and by lipid A-associated protein. J. Immunol. 118:1910-1911. 16. Landy, M., and P. J. Baker. 1966. Cytodynamics of the distinctive immune response produced in regional lymph nodes by salmonella somatic polysaccharide. J. Immunol. 97:670-679. 17. Landy, M, and W. Braun (ed.). 1964. Bacterial endotoxins. Rutgers University Press, New Brunswick, N.J. 18. McConahey, P. J., and F. J. Dixon. 1966. A method of trace iodination of proteins for immunologic studies. Int. Arch. Allergy 29:185-189. 19. Miner, K. D., J. A. Rudbach, and E. Ribi. 1971. General characteristics, p. 1-65. In G. Weinbaum, S. Kadis, and S. J. Ajl (ed.), Microbiological toxins, vol. IV. Academic Press Inc., New York. 20. Moeller, G. R., L. Terry, and R. Snyderman. 1978. The inammatory response and resistance to endotoxin in mice. J. Immunol. 120:116-123. 21. Moreau, S. C., and R. C. Slarnes. 1973. Host resistance to bacterial endotoxemia: mechanisms in endotoxin-tolerant animals. J. Infect. Dis. 128(Suppl.):S122. 22. Morrison, D. C., and C. G. Cochrane. 1974. Direct evidence for Hageman factor (factor XII) activation by bacterial lipopolysaccharides (endotoxins). J. Exp. Med. 140:797-811. 23. Morrison, D. C., and L F. Kline. 1977. Activation of the classical and properdin pathways of complement by bacterial lipopolysaccharides (LPS). J. Immunol. 118:363-368. 24. Morrison, D. C., and L Lieve. 1975. Fractions of lipopolysaccharide from Escherichia coli Olll:B4 prepared by two extraction procedures. J. Biol. Chem. 250:2911-2919. J. Shear. 1963. 25. Oroszlan, S. L, P. T. Mora, and Reversible inactivation of an endotoxin by intracellular protein. Biochem. Pharmacol. 12:1131-1146. 26. Parant, M., F. Parant, and L. Chedid. 1977. Inheritance of lipopolysaccharide-enhanced nonspecific resistance to infection and of susceptibility to endotoxic shock in

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16:432-438. 27. Reed, L J., and H. Muench. 1938. A simple method for estimating fifty percent endpoints. Am. J. Hyg.

27:493-497. 28. Reed, N. D., J. K. Manning, and J. A. Rudbach. 1973. Immunologic responses of mice to lipopolysaccharide from Escherichia coli. J. Infect. Dis. 128

(Suppl.):S70-S74.

29. Roos, D., and J. A. Loos. 1970. Changes in carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. I. Stimulation by phytohemmaglutinin. Biochem. Biophys. Acta 222:565-682. 30. Ruco, L. P., and M. S. Meltzer. 1978. Defective tumoricidal capacity of macrophages from C3H/HeJ mice. J.

Immunol. 120:329-334. 31. Rudbach, J. A. 1971. Molecular immunogenicity of bacterial lipopolysaccharide antigens: establishing a quantitative system. J. Immunol. 106:993-1001. 32. Rudbach, J. A., and A. G. Johnston. 1966. Alteration and restoration of endotoxin activity after complexing with plasma proteins. J. Bacteriol. 92:892-98. 33. Rudbach, J. A., and N. D. Reed. 1977. Immunological responses of mice to lipopolysaccharide: lack of secondary responsiveness to C3H/HeJ mice. Infect. Immun. 16:513-517. 34. Ryan, J. L., and K. P. W. J. McAdam. 1977. Genetic non-responsiveness of murine fibroblasts in bacterial endotoxin. Nature (London) 269:153-155. 35. Skidmore, B. J., J. M. Chiller, D. C. Morrison, and W. 0. Weigle. 1975. Immunologic properties of bacterial lipopolysaccharide (LPS): correlation between the mitogenic, adjuvant, and immunogenic activities. J. Immunol. 114:770-775. 36. Skidmore, B. J., J. M. Chiller, and W. 0. Weigle. 1977. Immunologic properties of bacterial lipopolysaccharide (LPS). IV. Cellular basis of the unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis. J. Immunol. 118:274-281. 37. Skidmore, B. J., D. C. Morrison, J. M. Chiller, and W. 0. Weigle. 1975. Immunologic properties of bacterial lipopolysaccharide. II. The unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS. J. Exp. Med. 142:1488-1508. 38. Sultzer, B. M. 1968. Genetic control of leukocyte responses to endotoxin. Nature (London) 219:1253-1254. 39. Sultzer, B. M 1969. Genetic factors in leukocyte responses to endotoxin: further studies in mice. J. Immunol. 103:32-38. 40. Sultzer, B. M. 1972. Genetic control of host responses to endotoxin. Infect. Immun. 5:107-113. 41. Sultzer, B. M., and G. W. Goodman. 1977. Characteristics of endotoxin-resistant low-responder mice, p. 304-309. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 42. Sultzer, B. M., and B. S. Nilsson. 1972. PPD-tuberculin-a B-cell mitogen. Nature (London) New Biol. 240:198-200. 43. Thomas, IL, and R. A. Good. 1952. Studies on the generalized Shwartzman reaction. I. General observations concerning the phenomenon. J. Exp. Med.

96:605-624. 44. Truffa-Bachi, P., J. G. Kaplan, and C. Bona. 1977. The mitogenic effect of lipopolysaccharide. Metabolic processing of lipopolysaccharide by mouse lymphocytes. Cell Immunol. 30:1-11. 45. Watson, J. 1977. Differentiation of B lymphocytes in C3H/HeJ mice: the induction of Ia antigens by lipopolysaccharide. J. Immunol. 118:1103-1108. 46. Watson, J., K. Kelly, M. Largen, and B. A. Taylor. 1978. The genetic mapping of a defective LPS gene in C3H/HeJ mice. J. Immunol. 120:422-424.

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VOL. 21, 1978 47. Watson, J., and R. Riblet. 1974. Genetic control of responses to bacterial lipopolysaccharides in mice. I.

Evidence for a single gene that influences mitogenic and immunogenic responses to lipopolysaccharides. J. Exp. Med. 140:1147-1161. 48. Watson, J., and R. Riblet. 1975. Genetic control of responses to bacterial lipopolysaccharides in mice. H. A gene that influences a membrane component involved in the activation of bone marrow-derived lymphocytes by lipopolysaccharides. J. Immunol. 114:1462-1468. 49. Watson, J., E. Trenkmer, and M. Cohn. 1973. The use

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of bacterial lipopolysaccharides to show that two signals required for the induction of antibody synthesis. J. Exp. Med. 138:699-714. 50. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides-extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-96. 51. Zlydaszyk, J. C., and R. J. Moon. 1976. Fate of 'Crlabeled lipopolysacchande in tissue culture cells and livers of normal mice. Infect. Immun. 14:100-105. are

Distribution of endotoxin (lipopolysaccharide) in the tissues of lipopolysaccharide-responsive and -unresponsive mice.

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