American Journal of Pathology, Vol. 138, No. 2, February 1991 Copright © American Association of Pathologis

In Vivo Biologic and Immunohistochemical Analysis of Interleukin-1 Alpha, Beta and Tumor Necrosis Factor During Experimental Endotoxemia Kinetics, Kupffer Cell Expression, and Glucocorticoid Effects

Stephen W. Chensue,*t Pauline D. Terebuh,* Daniel G. Remick,t Wendy E. Scales,t and Steven L. Kunkelt From the Departments of Pathology, Veterans Affairs Medical

Center* and the University of Michigan Hospitals, t Ann Arbor, Michigan

Using a model of sepsis induced by parenteral challenge of mice with bacterial lipopolysaccharride (LPS), the authors analyzed the in vivo expression of interleukin- I (IL- I) a, ,B and tumor necrosis factor (TNF). Both TNF and IL-Io, 1 were detected in hepatic sinusoidal macrophages (Kupffer cells), immunohistochemically. Kinetic analysis showed a clear sequence of synthesis. Tumor necrosis factor was produced first, reaching maximal expression at 1 hour after LPS challenge, then rapidly disappeared IL- 1,B followed, reaching maximal expression at 2 to 3 hours, then dropped off by 6 hours. Interleukin-la expression reached a peak at 6 hours and had disappeared by 18 hours. Analysis of serum bioactivity also revealed sequential expression that correlated with immunohistochemical findings. Tumor necrosis factor was maximal at I hour and IL-I at 6 hours. The IL-I bioactivity was not due to interleukin-6 (IL6), as this was depleted from specimens by immunoabsorption. Also IL-6 bioactivity reached maximal levels at3 hours, earlier than IL-I. Pretreatment with 4 mg/kg dexamethasone significantly decreased Kupffer cell expression of TNF and IL-I a (about 80% and 60% suppression, respectively) but had less effect

IL-13 expression (about 30% suppression). Accordingly, serum levels of TNF were suppressed by 75% while serum IL-I was decreased by 39%, indicating differential sensitivity of these cytokines to

on

glucocorticoids. Endogenous corticosteroid levels increased as TNF levels decreased, supporting the contention that glucocorticoids regulate TNF synthesis. In contrast, IL-I levels rose concurrently with corticosterone. These data indicate a sequential activation of cytokine gene expression in vivo, which may be critical to the cascade of events leading to septic shock, and provide evidence that Kupffer cells are a major source of cytokines in endotoxemia Finally, the differential sensitivity of cytokine expression to glucocorticoids may in part explain the inadequacy of the latter in the treatment of sepsis. (Am JPathol 1991, 138:395-402)

There is growing evidence that the pathophysiologic manifestations of many disease states is due to the production of a variety of polypeptide products known as cytokines (eg, interferons, interleukins, and tumor necrosis factors). Major sources of these molecules are leukocytes of the immune system, but other tissues can likely produce them. Under physiologic conditions, these molecules are thought to be important in regulating the activity of immune and stromal cells. When produced aberrantly or in excess, however, they can have toxic effects. There is strong evidence that the clinical manifestations of septic shock are due to such aberrant production of cytokines, especially interleukin-1 (IL-1) and tumor necrosis factor (TNF).1" Sessile macrophages such as Kupffer Supported by the Department of Veterans Affairs and NIH grants HL31237, HL31963, and HL35276. Dr. Kunkel is an Established Investigator of the American Heart Association. Accepted for publication October 1, 1990. Address reprint requests to Dr. Stephen Chensue, Department of Pathology Laboratory SVC 113, VAMC, 2215 Fuller Rd, Ann Arbor, MI 48105.

395

396

Chensue et al

AJP Febnuiay 1991, Vol. 138, No. 2

cells of hepatic sinusoids are postulated to be a source of these cytokines, but there is little direct evidence showing this in vivo.7 Using a model of sepsis induced by parenteral endotoxin challenge, we have analyzed the in vivo production of TNF and IL-1. We demonstrated the expression of TNF, IL-1a, and IL-1 in Kupffer cells by immunohistochemical means. There was a distinct sequential appearance of cytokine expression that correlated with the appearance of bioactive material in the serum. The sequence of production was similar to that described in baboon and rabbit models.8'9 Production and cytoplasmic expression of IL-1 and TNF could be reduced by dexamethasone pretreatment, but the latter to a greater extent than the former, indicating that cytokines have different sensitivities to dexamethasone administered in vivo. These studies have important implications regarding the production and regulation of cytokines during acute pathologic states.

Materials and Methods Animals Female, CBA/J or NIH Swiss albino outbred mice were used for experiments. Mice were maintained under specific pathogen-free conditions and provided with food and water ad libitum.

Endotoxin Treatment and Tissue Preparation Mice were given 80 ,ug lipopolysaccharide (Escherichia coli 0111 ) intraperitoneally in a 0.8-ml volume. At specified intervals, the mice were exsanguinated and their livers were excised, placed in tissue-mounting fluid, and snap frozen in liquid nitrogen. Tissues were stored at -800C.

Dexamethasone Treatment Dexamethasone solutions were prepared just before use. Solid material was dissolved at 10 mg/ml in ethanol and then diluted in saline before injection. Each animal was administered 4 mg/kg by intraperitoneal injection 3 hours before endotoxin challenge.

Antibodies Goat anti-IL-1 a and rabbit anti-IL-1 ,B and anti-TNF ax were produced by immunization with recombinant pro-

teins administered in multiple intradermal sites with Freund's complete adjuvant. The anti-TNF has high titer (1:1000) activity in neutralizing bioactive material and in immunoperoxidase staining of in situ material. The antiIL-1 a and ,B reacted with 30-kd cytoplasmic precursors as well as 17-kd processed molecules in Western blot assays. Both also inactivated bioactivity of the respective recombinant molecules and had high titer (1:1000 and 1:2000, respectively) in immunolocalization studies. Specificity, of these polyclonal preparations in immunolocalization has been confirmed by competitive inhibition and immunoabsorption and previously reported.10 12 Goat anti-murine interleukin-6 (IL-6) was provided by Dr 1. Otterness, Pfizer Central Research (Groton, CT). This preparation showed no cross-reactivity with TNF or the two forms of IL-1. Monoclonal rat anti-mouse MAC-2 (Hybritech Inc, San Diego, CA) was used at 25 ,ug/ml to localize hepatic macrophages.

Immunohistochemistry Five- to seven-micron-thick frozen sections were prepared and mounted on poly-L-lysine-coated glass slides. The mounted tissues were immediately fixed for 10 minutes in cold acetone. The slides were next rehydrated in PBS (phosphate-buffered saline) and treated with avidin and biotin to block binding sites for these molecules. Next they were treated with a 1:50 dilution of blocking serum for 10 minutes at 37C and then exposed to optimal dilutions of specific antibody and similar dilutions of control sera. After 20 minutes' incubation at 370C, the slides were rinsed thrice with PBS, overlaid with biotinylated secondary antibodies (1 :100) (Vector Laboratories), and incubated another 10 minutes, followed by three additional rinses with PBS. At this point, sections were treated with alkaline phosphatase-labeled streptavidin, rinsed thrice, then overlaid with substrate chromogen (Vector Laboratories) and incubated for 25 min at room temperature to allow for color development. Mayer's hematoxylin was used as a counterstain. Slides were coded and evaluated blindly.

Cytokine Assays Tumor necrosis bioactivity was measured with the highly sensitive cell line, WEHI 164 subclone 13, provided by Dr. Anders Waage, University of Trondheim, Norway. 13 The target cells are highly sensitive to the cytolytic activity of TNF, detecting as little as 2 pg/ml. The cells are uneffected by other cytokines such as y-interferon or IL-1. 14 Briefly, samples are serially diluted in 96-well culture dishes using Roswell Park Memorial Institute (RPMI) me-

Cytokine Expression In Vivo

397

AJP Feb&ruy 1991, Vol. 138, No. 2

dium with 1% fetal bovine serum (FBS) as a diluent. WEHI 164 cells suspended in RPMI with 10% FBS, 1 mmol/l [millimolar] glutamine, and 0.5 ,g/ml actinomycin D (Calbiochem, La Jolla, CA) are added to each well, then incubated overnight. Viability is determined the following day with MTT tetrazolium. The plates are read using a microELISA reader and units of activity are determined from a standard curve. Interleukin-1 bioactivity was determined in IL6-depleted sera using the standard thymocyte costimulation assay modified from the procedure of Mizel et al.15 and Koopman et al.16 Thymocyte suspensions were prepared from 5- to 7-week-old CBA/J mice, washed twice with RPMI-1640, and resuspended to a density of 5 x 106 cells/ml in RPMI-FBS. Thymocytes (5 x 1 05 per well) were cultured for 72 hours in 0.2 ml of RPMI (with 10% FBS, 1 mmol/l glutamine, 100 ,ulml penicillin and 100 ,ug/ml streptomycin) in 96-well flat bottom culture plates (Costar, Cambridge, MA) in the presence or absence of 1 ug/ml purified PHA (Wellcome Research Labs, Beckenham, England) and containing 1092 dilutions of serum (starting dilution was 1:10). Each culture well was pulsed with 1.0 ,uCi 3H-methyl-thymidine (1.9 Ci/mmole, ICN, Irvine, CA) for the final 6 hours of incubation. Cells are then collected on glass fiber filter strips with an automatic cell harvester (Cambridge Technology, Inc., Watertown, MA). Standards were recombinant murine IL-1a and P. Essentially identical standard curves were generated by the two forms. Interleukin-6 bioactivity was determined by the standard B9 cell proliferation assay.17 The cells were provided by Dr. J. Gauldie, McMaster University, Hamilton, Ontario. Proliferation was assessed at 72 hours with MTT as a colorimetric indicator.

Immunoabsorption Assessment of IL-1 bioactivity in sera is difficult because of presence of potential inhibitors as well as IL-6, which can drive the thymocyte proliferation assay.18 To minimize these interfering agents, sera to be assayed for IL-1 activity was first passed through an anti-murine IL-6 immunoaffinity column. The columns were prepared and used as previously described.12 Immunoabsorption appeared to be specific, as both TNF and IL-1 activity were unaffected by column passage. Column passage reduced IL-6 activity by about 99% compared to the control immunoabsorbent. The column passed sera was next dialyzed overnight to remove low molecular weight IL-1 inhibitors. In addition, the preparations were assayed at final dilutions of 1:20, 1:40, and 1:80 to minimize interference by high molecular weight inhibitors.

Corticosterone Determination Plasma corticosterone levels were determined by a licensed independent clinical laboratory by radioimmunoassay (Mayo Medical Laboratories, Rochester, MN). Corticosterone represents the predominant glucocorticoid produced by mice.

Statistics The Student's t-test was used to compare control and experimental groups. Values of P < 0.05 were considered to indicate lack of significance.

Results Distribution and Kinetics of Cytokine Staining After Endotoxin Challenge Figure 1 shows the staining for TNF, IL-1a, and IL-1 in livers of mice at 1, 6, and 3 hours after endotoxin challenge, respectively. Staining for all of the cytokines was clearly restricted to sinusoidal lining cells and was not observed in hepatocytes, ductal cells or endothelial cells. Morphologically, the staining cells were consistent with sessile macrophages (Kupffer cells) and the staining distribution was identical to that for the Mac-2 (mouse macrophage) antigen marker. Staining was not observed in unchallenged mice. Interestingly the patterns of staining for TNF and IL-1 within cells was different. Interleukin-1 staining was generally diffusely cytoplasmic, whereas TNF staining tended to be more localized within the cytoplasm. Figures 2 and 3 show the kinetics of cytokine expression in sinusoidal macrophages. TNF was consistently the first of the cytokines to be expressed, reaching maximal expression at 1 hour after challenge, then rapidly disappeared, being nearly undectable by 3 hours. IL-1 3 appeared immediately after, reaching maximal expression at 2 to 3 hours. In turn, IL-1 a expression followed and was more prolonged, reaching a maximum from 3 to 6 hours. Both forms of IL-1 were no longer detected by 18 hours. Thus, there was a clear sequence of cytokine expression in macrophages in vivo after endotoxin challenge. Furthermore essentially identical results were obtained in both inbred and outbred mice.

Kinetics of TNF, IL-6, and IL-1 Bioactivity in Sera After Endotoxin Challenge It was important to establish the relationship between cytokine expression detected immunohistochemically and

398

Chensue et al

AJP Febrwary 1991, Vol. 138, No. 2

Figure 1. Immunobistochemical stain of cytokines in livers of endotoxin challenged CBA mice. A: anti-murine IL-la, 6 hours after LPS challenge; B: anti-murine IL-I1 , 3 hours after LPS challenge; C: anti-murine TNFa, 1 hour after LPS challenge; D: nonimmune serum control (magnification 400X; insets, 100OX).

the presence of biologically active material. As shown in Figure 4, the presence of biologic activity in the serum paralleled closely immunohistochemical expression. Serum TNF activity was maximal at 1 hour and was undetectable by 4 hours. This decline was not due to neutral-

izing factors such as circulating TNF receptors, since the sera did not affect the activity of added recombinant TNF. Figure 5 shows the appearance of IL-1 and IL-6 bioactivity in sera after lipopolysaccharride (LPS) challenge. Interleukin-1 activity was measured in IL-6-depleted sera

Cytokine Expression In Vivo

399

AJP Februay 1991, Vol. 138, No. 2

aLX

a-

-J C,)

(A

l0J 0

0-

I

0 2 4 6 8 1012141618202224 HOURS Figure 2. Time course of TNF expression in bepatic sinusoidal macrophages of endotoxin-challenged CBA mice. Points are means + SEM of three separate experiments.

to avoid interference by this cytokine.18 Interleukin-1 appeared at 3 hours, was maximal at 6 hours (4.4 ng/ml), and appeared to partially drop off by 18 hours. Interleukin-6 activity sharply increased to high concentrations (132 ng/ml) by 3 hours then fell off rapidly over the next 3 hours, finally reaching basal levels by 18 hours. Thus IL-6 was clearly separable from IL-1 activity. It should be noted that despite depletion of IL-6, the possible contribution of other factors with IL-1 activity cannot be eliminated. In preliminary immunoabsorption studies, however, the serum IL-1 activity appears to be largely IL-1 P. In summary, there was close correlation of immunohistochemical and biologic findings, indicating an orchestrated sequence of cytokine release.

Role of Glucocorticoids in the Regulation of Cytokine Expression In Vivo We and others have previously shown that corticosteroids can suppress TNF expression in recruited peritone-

HOURS Figure 4. Time course of appearance of serum TNF bioactiit* in endotoxmn-challenged NIH swiss mice. Points are means + SEM of three to five mice.

al macrophages in vivo and in vitro.192' Likewise, in vitro studies show corticosteroids to suppress IL-1 expression.2122 It was of interest to determine the relationship of glucocorticoids to Kupffer cell cytokine expression. Mice were treated with 4 mg/kg dexamethasone 3 hours before endotoxin challenge, then Kupffer cell expression of TNF and IL-1 was examined at 1 and 3 hours after challenge. Figure 6 shows the effect of dexamethasone on Kupffer cell cytokine expression. The number of cells with TNF expression was suppressed by about 80% (P < 0.001). Similarly IL-1 a expression was suppressed by nearly 60% (P < 0.001). Interestingly IL-1 P seemed more resistant to treatment and was suppressed by only 30% (P < 0.05). Thus corticosteroid treatment was not equally effective in blocking the cytoplasmic expression of these cytokines. As shown in Table 1, levels of serum bioactivity for TNF and IL-1 reflected the suppression observed immunohistochemically. Tumor necrosis factor production was most sensitive to dexamethasone, being decreased by 75%, while IL-1 activity was abrogated by about 39%. Suppression was not due to the presence of dexameth150

IL

125 hJ CL 1001oo

cn

75 LU

0

-j

t=

50

0 0-

-g

C LO n

25

0 2 4 6 8 10 12 14 16 18 20 22 24 HOURS Figure 3. Time course of IL-la and (3 expression in hepatic sinusoidal macrophages ofendotoxin-challenged CBA mice. Points are means + SEM of three separate experiments.

o 0

2

4

6

8

10

12. 14

16

18 20

HOURS Figure 5. Time course of appearance of serum IL-1 and IL-6 bioactivity in endotoxin-challenged NIH sws mice. Points are means + SEM of three to five mice.

400

Chensue et al

AJP Febnrary 1991, Vol. 138, No. 2

.,

14

Liii

r,II

12

E

-J _-LJ bJ

10

E

CONTROL

LL

DEXAMETHASONE

LLI

w

z 0io w

a

C0 c

Li 4

CL

2 0

I

r--------

IL- 1 alpha

0 r

ro

IL- 1 beta

rql vf

e

TNF alpha

Figure 6. Effect of dexamethasone treatment on cytokine expression in hepatic sinusoidal macrophages in endotoxin-challenged NIH swiss mice. Bars are means ± SEM of three separate eperiments.

asone in the assay system, as this agent has no effect on the WEHI assay for TNF up to 10-5 molA (molar) concentiations. Similarly dexamethasone levels present in treated mice were not sufficient to affect the IL-1 assay, because pooled sera of dexamethasone-treated mice did not affect the detection of exogenously added recombinant IL-1 (data not shown). Endotoxemia is known to induce the endogenous production of glucocorticoids. Furthermore both TNF and IL1 have been reported to induce cortisol production when administered in vivo.23'24 To further explore the relationship of glucocorticoids to cytokine production, we measured endogenous corticosterone levels in LPSchallenged mice. Untreated animals were killed in parallel to control for diurnal changes in glucocorticoid levels (see legend, Figure 7). As shown in Figure 7, LPS caused a rise in corticosterone levels by 1 hour that became maximal at 3 to 6 hours after challenge. By 24 hours, levels had significantly declined but were still above controls. Interestingly glucocorticoids were increased concurrently with serum IL-1 levels (Figure 7). Hence there appeared to be a teleologic basis for our observation that IL-1 was more resistant to the effect of administered dexamethasone, because this cytokine was normally produced in a glucocorticoid-rich environment during the physiologic response to endotoxin. Table 1. Effect of Dexamethasone on Serum Cytokine Levels Concentration (ng/ml ± SD)* Percentage Control Dexamethasone Cytokine change -75% TNF 59.3 + 12.6 14.9 + 0.01 (P < 0.005) 4.8 ± 2.0 2.9 ± 0.62 -39% IL-1t (P < 0.025) Cytokine levels are reported at the times of expected maximal expression, TNF at 1 hour and IL-1 at 6 hours. t IL-1 levels were determined after depletion of IL-6 by immunoabsorbent; IL-6 was depleted from 52.0 to 0.45 ng/ml.

(9

500 450 400 350 300 250 200 150 100 50 0

1rD 9

8 7 6 5 4 3 2

11.1-1.1.,..,-,-,.,..'..................................................A

E Cn 171 D

C/)

.................................

0

1

3 HOURS

6

0

24

Figure 7. Time course of serum corticosterone appearance in endotoxin-challenged NIH swiss mice in relation to IL-i production. Equal volumes ofplasma were pooled from a minimum of three mice then assayedfor corticosterone. Levels in control groups at 1, 3, and 6 hours were 24, 72, and 120 ng/ml, respectively.

Discussion To date, numerous polypeptide cytokines have been identified with a plethora of endocrine, paracrine, and autocrine functions with potentially important roles in disease.25 Many of these functions have been defined in vitro; therefore, the current challenge is to define their precise role in vivo. We have previously used immunohistochemical and biologic methods to analyze cytokine production in cultured macrophages.10' In the present study, we applied this approach to analyze production in vivo. Specifically, we examined the production of tumor necrosis factor (TNF), IL-6 and the a and a forms of IL-1, during experimentally induced endotoxemia. These cytokines are thought to play an important role in the pathologic manifestations of sepsis/endotoxemia.1-6 Furthermore, it has been postulated that cells of the mononuclear phagocytic system (MPS) are a major source of these polypeptide mediators.7 Our study provides several novel findings with regard to these questions. We have shown immunohistochemically the in vivo appearance of cytokines in response to endotoxin challenge. Specifically, TNF and IL-1 were demonstrated within hepatic sinusoidal macrophages (Kupffer cells), supporting the notion that cells of the MPS are indeed important sources of cytokines. It is noteworthy that the patterns of focal cytoplasmic staining for TNF and diffuse staining for IL-1 are identical to those that we observed in cultured macrophages challenged with endotoxin.26 At present, we have been unable to detect IL-6 immunohistochemically in Kupffer cells after intraperitoneal endotoxin challenge. It is not known if this indicates an inability to produce the cytokine or simply reflects the specific experimental conditions. For example, intravenous challenge may be required to demonstrate expression. Initial studies suggest IL-6 expression in vivo is complex, being produced by a variety of cell types. A detailed anal-

Cytokine Expression In Vivo

401

AJP Febnuy 1991, Vol. 138, No. 2

ysis of the distribution of IL-6 expression is underway and will be reported later. The present study clearly demonstrates TNF and IL-1 expression in Kupffer cells. Initial studies in our laboratory have shown similar findings for sessile macrophages of the lung, suggesting that cells of the MPS are indeed major sources of these cytokines. Other cells such as endothelial cells and circulating monocytes are also potential sources. In the mouse, however, we have not seen positive staining in endothelial cells in vivo. In our model, circulating monocytes are likely not a major source of cytokines, because they normally constitute a minor component (