Lipopolysaccharide (LPS) binding to 73-kDa and 38-kDa surface proteins on lymphoreticular cells: preferential inhibition of LPS binding to the former by Rhodopseudomonas sphaeroides lipid A

Immunology Letters, 36 (1993) 245-250 0165 - 2478 / 93 / $ 6.00 ,© 1993 ElsevierSciencePublishers B.V. All rights reserved

IMLET 01974

Lipopolysaccharide (LPS) binding to 73-kDa and 38-kDa surface proteins on lymphoreticular cells: preferential inhibition of LPS binding to the former by Rhodopseudomonas sphaeroides lipid A M e i - G u e y Lei a, N i l o f e r Q u r e s h i b a n d D a v i d C. M o r r i s o n a'c ~Department of Microbiology, Molecular Genetics and Immunology, and CThe Cancer Center, University of Kansas Medical Center, Kansas City, KS 66160, USA; and bMycobacteriology Laboratory, William S. Middleton Memorial Veterans Hospital, Madison, W153705, USA, and Department of Bacteriology, College of Agricultural Life Science, University of Wisconsin, Madison, W153706, USA

(Received 27 January 1993; accepted 27 March 1993)

I.

Summary

Using a photoactivable, radioiodinated lipopolysaccharide probe, [125I]ASD-LPS (derivatized from purified E. coli 011 l:B4 S-LPS), we earlier reported the presence of a 73-kDa (p73) predominant LPS-binding protein on mouse lymphocytes and macrophages with specificity for the lipid A region of LPS. Both Re-LPS from Salmonella minnesota and purified lipid A will inhibit the binding of LPS to the p73 LPS receptor. In the studies reported here, we have found that nontoxic diphosphoryl lipid A purified from Rhodopseudomonas sphaeroides has the capability to inhibit the binding of [125I]ASD-LPS to the p73 protein. However, using the same LPS probe and photoaffinity cross-linking techniques, our data suggest that a less dominant 38-kDa (p38) LPSspecific binding protein identified on mouse splenocytes, J774.1 macrophage-like cell line, and 70Z/3 pre B-cell line by SDS-PAGE is not inhibited by purified lipid A, even at a concentration in 50-fold excess of that of [~25I]ASD-LPS. The binding of the LPS probe to the p38 protein Key words." LPS-binding protein; Kdo determinant; Lipid A;

Rs-DPLA Correspondence to." Dr. M.-G. Lei, Department of Microbiology, MolecularGenetics and Immunology,Universityof Kansas Medical Center, Kansas City, KS 66160, USA.

could be inhibited in a dose-dependent manner by underivatized native S. minnesota Re-LPS (composed only of Kdo and lipid A). We speculate that this p38 LPS-binding protein may manifest a specificity for inner core oligosaccharide determinants on LPS. 2.

Introduction

We have previously identified a membrane-localized p73 LPS-binding protein on mouse lymphoreticular cells [1-3]. More recently, we have demonstrated the existence of similar p73 LPSbinding proteins on peripheral blood mononuclear cells of a variety of endotoxin-sensitive mammalian species, including man [4,5]. We have reported that the binding of LPS to the p73 protein was inhibitable by lipid A purified from S. minnesota Re-LPS [1]. We have also shown that a monoclonal antibody MAb5D3 raised against the mouse p73 LPS-binding receptor could activate mouse macrophages for tumor cell killing [6] and nitric oxide production [7] in vitro and protect mice against the lethal effect of LPS in vivo [8]. Collectively, these data provide support for the concept that p73 can serve as a functional receptor for LPS in triggering lymphoreticular cell responses. Highly purified pentaacyl diphosphoryl lipid A derived from the LPS of R. sphaeroides (RsDPLA) has been shown to inhibit T N F and IL-1 245

production in macrophages stimulated by endotoxic Escherichia coli Re-LPS in vitro [9,10] and also to inhibit the transient rise of circulating T N F in mice in response to Re-LPS injection in vivo [10]. These results are consistent with RsDPLA functioning as a receptor antagonist [11]. Experiments were therefore carried out to determine whether this Rs-DPLA could inhibit the binding of LPS to the p73 protein. In the course of these studies, a less dominant but nevertheless still prominent p38 LPS-binding protein was identified on mouse splenocytes, 70Z/3 pre-B-cell line, and the J774.1 macrophage-like cell line. Whereas the binding of [125I]ASD-LPS to the p73 protein can be inhibited efficiently with S-LPS and ReLPS as well as with both wild-type lipid A and the lipid A derived from R. sphaeroides, only the first two inhibitors were effective in inhibiting LPS binding to the p38 protein. Since Re-LPS is composed only of the oligosaccharide Kdo and lipid A, these data suggest that the p38 LPS-binding protein may have a specificity for the inner core Kdo determinants on the LPS molecule. 3.

Materials and Methods

3.1.

Reagents

LPS from E. coli 0111:B4 was extracted and purified by the phenol-water procedure of Westphal and Luderitz [12], as previously described [13]. Re-LPS from S. minnesota R595 was extracted by the phenol-chloroform-petroleum ether method of Galanos et al. [14]. Purified lipid A from S. minnesota was purchased from List Biological Laboratories (Campbell, CA). RsDPLA from R. sphaeroides was purified as described previously [15]. Sulfosuccinimidyl-2-(pazidosalicylamido)- 1,Y-dithiopropionate (SASD) was purchased from Pierce Chemical Co. (Rockford, IL). Photoactivable, iodinated E. coli 0111:B4 LPS ([125I]ASD-LPS, approximate specific activity of 2/~Ci/#g) was prepared by radioiodination of the SASD-conjugated LPS exactly as described previously [16]. 3.2.

Mice

Mice of the C3HeB/FeJ strain were purchased 246

from Jackson Laboratory (Bar Harbor, ME) and maintained in the Animal Facility at the University of Kansas Medical Center until used for experiments. Mice used were between 2 and 3 months of age and were of either sex. 3.3.

Splenocytes and cell lines

Mouse spleen cells were prepared as described elsewhere [17]. Red blood cells were lysed by hypotonic shock procedure as described by Mishell and Shiigi [18]. After lysis of red blood cells, spleen cells were washed three times with cold medium (RPMI 1640) and by centrifugation at 300 x g. The mouse pre-B-cell line 70Z/3 was obtained from the American Type Culture Collection (Washington, DC) and cultured as described previously [3]. The mouse macrophage-like cell line J774.1 was kindly provided by Dr. T. Suzuki (Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center). This cell line was maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 /~g/ml streptomycin. Cell cultures were washed three times with cold RPMI medium before being used for the photoaffinity labeling. 3.4.

Photoaffi'nity labeling

Photoaffinity labeling was carried out as described previously [3]. Briefly, cells (2 x 10 6 splenocytes, 1 × 106 70Z/3 cells, or 5 x 105 J774.1 cells) were incubated with [125I]ASD-LPS (1 #g LPS) at 37°C for 30 rain (unless otherwise indicated). After incubation, the reaction mixture was UV-irradiated for 10 min to effect photocrosslinking of LPS to the target molecules. Cells were washed three times with cold medium, lgsed by using SDS sample buffer in the presence of 2ME, boiled for 5 min, and applied to SDS-PAQE analysis. 3.5.

SDS polyacrylamide-gel electrophoresis

One-dimensional 11% SDS-PAGE was performed as described by Laemmli [19]. Molecular weight markers were purchased from Bio-Rad

( R i c h m o n d , CA). Gels were stained, destained, dried, a n d a u t o r a d i o g r a p h e d to detect L P S - b i n d ing molecules as d e s c r i b e d p r e v i o u s l y [3].

4.

Results and Discussion

To determine whether the nontoxic lipid A preparation, R s - D P L A , w o u l d b i n d to the p73 L P S receptor, e x p e r i m e n t s were carried o u t in which an excess a m o u n t o f this lipid A was e x a m i n e d for its c a p a b i l i t y to inhibit the p h o t o a f f i n i t y labeling o f the p73 r e c e p t o r on m o u s e splenocytes by [125I]ASD-LPS. W e have p r e v i o u s l y d e m o n s t r a t e d t h a t b o t h R e - L P S a n d lipid A will inhibit L P S b i n d i n g to the p73 r e c e p t o r [1,3]. In the cur-

97ep73

66-

rent study, R e - L P S was used as a positive control. The results o f one such e x p e r i m e n t are shown in Fig. 1. R s - D P L A manifests a similar ability to R e - L P S in inhibiting [~25I]ASD-LPS b i n d i n g to the p73 r e c e p t o r (lanes 4 a n d 5 vs. lanes 2 a n d 3). T h e use o f d e n s i t o m e t r i c s c a n n i n g o f the intensities o f the p73 b a n d s on the a u t o r a d i o g r a p h has allowed a m o r e q u a n t i t a t i v e e s t i m a t i o n o f the inhibition. In the presence o f 50- or 100-fold excess o f R s - D P L A (lanes 4 a n d 5, respectively), the intensity o f the a u t o r a d i o g r a p h o f the p73 r e c e p t o r was r e d u c e d to 4 1 % a n d 2 8 % , respectively, as c o m p a r e d with the c o n t r o l in the absence o f inhib i t o r (lane 1). These inhibition d a t a are similar to

9766-

~p73

43<--p38

43~p38 31312121-

14-

141

2

3

4

5

Fig. l. SDS-PAGE analysis of [t25I]ASD-LPS photoaffinitylabeled mouse splenocytes in the presence of underivatized ReLPS or Rs-DPLA. Inhibitors were added to the photoaffinity labeling incubation mixture at the same time as [1251]ASD-LPS. Lane 1, control, without inhibitor; lanes 2 and 3, plus 50 and 100 ,ug of S. minnesota Re-LPS, respectively; lanes 4 and 5, plus 50 and 100 #g of Rs-DPLA derived from R. sphaeroides, respectively. In each lane, 2 x 1 0 6 cells were loaded. The result shows the autoradiograph of the SDS-PAGE.

1

2

3

4

5

Fig. 2. SDS-PAGE analysis of [~251]ASD-LPS photoaffinitylabeled mouse 70Z/3 pre-B-cell line in the presence of underivatized Re-LPS or Rs-DPLA. Inhibitors were added to the photoaffinity labeling incubation mixture at the same time as [~25I]ASD-LPS. Lane 1, control, without inhibitor; lanes 2 and 3, plus 50- and 100-fold excess of S. minnesota Re-LPS, respectively; lanes 4 and 5, plus 50- and 100-fold excess of R. sphaeroides Rs-DPLA, respectively. In each lane, 1 x l 0 6 cells were loaded. The result shows the autoradiograph of the SDSPAGE. 247

TABLE 1 Binding of [125I]ASD-LPS to the 38-kDa LPS-binding protein on mouse splenocytes and 70Z/3 pre-B-cell line in the presence of excess underivatized S. minnesota Re-LPS and R. sphaeroides Rs-DPLA.a Inhibitor

Percent of control b

Re-LPS 50 x Re-LPS 100x Rs-DPLA 50x R s - D P L A 100 x

Mouse splenocytes

70Z/3 pre B-cells

20.8% 8.1% 70.0% 48.8%

5.2% 0% 78.1% 49.3%

aDensitometric measurements from Figs. l and 2. bThe peak areas of the control without the inhibitor were 100% binding.

those observed in the presence of 50- or 100-fold excess of Re-LPS (lanes 2 and 3, respectively), in which the binding of the LPS probe to the p73 receptor was reduced to 39% and 21% of the control, respectively.

We further tested the ability of Rs-DPLA to inhibit [125I]ASD-LPS binding to the mouse 70Z/ 3 cell line. Similar results were obtained and are shown in Fig. 2. In the presence of 50- or 100fold excess of Rs-DPLA (lanes 4 and 5, respectively), the binding of [125I]ASD-LPS to the p73 receptor was reduced to 24% and 13%, respectively, as compared with the control in lane 1 in the absence of the inhibitor. In the presence of 50- or 100-fold excess of Re-LPS (lanes 2 and 3), the binding of the LPS probe to the p73 receptor was reduced to 33% and 9% of the control, respectively. These data are consistent with the hypothesis that the Rs-DPLA manifests equivalent activity to Re-LPS in inhibiting the binding of LPS to the p73 LPS receptor. In the course of analyzing the inhibition profiles for binding to p73 shown in Figs. 1 and 2, we noted that the binding of [125I]ASD-LPS to a second LPS-binding protein, which has a relative molecular mass of 38 kDa (p38), on both splenocytes and 70Z/3 cells could be effectively inhibited

PERCENTOFCONTROL

PERCENTOFCONTROL 0

20

40

60

I

r

I

80 100 I

20 40 60 80 100 I

I

CONTROL

CONTROL

Re-LPS 5X

Re-LPS

2ox

20X

5OX I

50X I

LIPID~X

i

t

i

i

140 i

I

5X

m

LIPIDA 5X

III

20X 20X i 50X S-LPffox

50X

II

S-LPS ! 5OX

Fig. 3. Binding of [125I]ASD-LPS to the 73-kDa and 38-kDa LPS-binding proteins on mouse splenocytes in the presence of excess underivatized S-LPS, Re-LPS, and purified lipid A. Samples were analyzed by SDS-PAGE and autoradiography. The corresponding p73 and p38 protein bands detected on the autoradiographs were scanned with a laser scanning densitometer (Biomed Instruments). The peak areas of the control without the inhibitor were 100% binding.

248

by Re-LPS, but significantly less so by Rs-DPLA (Table 1). We therefore compared the relative binding of [~25I]ASD-LPS to both the p73 and p38 proteins on splenocytes in the presence of excess underivatized S-LPS, Re-LPS, and lipid A. The experimental protocol employed was essentially identical to that described above. Quantitative determinations of [~25I]ASD-LPS binding to both p73 and p38 were assessed by densitometry of the autoradiographs. The results of one experiment are shown in Fig. 3 and provide support for the conclusion that while S-LPS and Re-LPS efficiently inhibited the binding of [125I]ASD-LPS to both p73 and p38 proteins, the purified lipid A only inhibited the binding to the p73 receptor and not to the p38 protein. These results are consistent with those reported previously [1-3] and with the data shown in Figs. 1 and 2, demonstrating that the p73 LPS receptor has a specificity for the lipid A region of the LPS molecule. More importantly, these data further suggest that this

A

B

97-

97-

66-

~p73

~-p73

66-

43-

43-

~-p38

~-p38

31-

31-

21-

21-

14-

14-

1

2

1

2

Fig. 4. Binding of [~251]ASD-LPSto the p38 protein. (A) Splenocytes. Lanes 1 and 2, incubation at 37°C and 0°C, respectively, and 2 x 10 6 cells were loaded on each lane. (B) J774.1 macrophage-like cell line. Lanes 1 and 2, incubation at 37°C and 0°C, respectively,and 5 × 105 cells were loaded on each lane.

newly identified p38 LPS-binding protein may have a specificity for the inner core region Kdo determinants of the LPS molecule (Re-LPS is composed of Kdo and lipid A). Since mouse macrophages have also been demonstrated to express the p73 LPS receptor independently of the temperature, we carried out experiments to assess whether the p38 LPS-binding protein can be detected on these cells and whether such binding is also temperature independent. For these studies, the J774.1 macrophagelike cell line and mouse splenocytes were incubated with [~25I]ASD-LPS at either 37°C or 0°C. Fig. 4 shows the identification of the p38 protein either at 37°C or 0°C on both splenocytes (Fig. 4A, lanes 1 and 2, respectively) and the J774.1 cell line (Fig. 4B, lanes 1 and 2, respectively). The results suggest that the binding of LPS to the p38 protein is also temperature-independent. Our experiments also allowed the detection of LPS-binding proteins in addition to those with an apparent molecular mass of 73 kDa and 38 kDa. Particularly noteworthy are proteins of about 50 kDa and 18 kDa. Similar proteins were identified earlier both in our laboratory [4,5] and by others [20-22]. The characteristics of these LPS-binding proteins are still under investigation in our laboratory. The fact that these proteins are detectable in the presence of protease inhibitors suggested that these proteins are probably not the result of degradation by proteases during the photocrosslinking procedure [5]. It will be of interest to identify these proteins in future studies. Collectively, these results suggest that at least two independent LPS-binding proteins exist on the surface of mouse lymphoreticular cells, a protein of approximately 73 kDa which has a specificity for lipid A and may function as a signal transducing molecule, and a second protein of 38 kDa which may have a specificity for non-lipid-A inner core oligosaccharide determinants. The ability of the nontoxic Rs-DPLA to inhibit the binding of LPS to p73 is consistent with its biological role of inhibiting lipid-A-dependent LPS responses in vitro and in vivo. The function of the p38 LPS-binding protein is, at present, unclear. It is noteworthy, however, that several recent studies have suggested that Kdo itself may activate both human and mouse macrophages for IL-1 produc249

tion [23,24] and that Kdo may be necessary for the full activity of LPS in stimulating mouse macrophages to prostaglandin and leukotriene production [25]. Further, a recent abstract has suggested the existence of specific inner core LPS-binding sites on hepatocytes [26]. Future studies will address the potential functional role of this newly identified p38 inner-core-specific LPSbinding protein.

[9] [10] [l 1]

[12] [13]

Acknowledgements [14]

This work was supported by research grants R37-AI-23447, PO1-CA54474 and GM-36054 from the National Institutes of Health. This work was also supported in part by the Medical Research Service, Department of Veterans and Affairs.

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