lymphocytes. metabolism in antigen-binding B ...

Antigen-induced changes in phospholipid metabolism in antigen-binding B lymphocytes. C D Myers, M K Kriz, T J Sullivan and E S Vitetta This information is current as of August 24, 2017.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1987 by American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 1987; 138:1705-1711; ; http://www.jimmunol.org/content/138/6/1705

0022-1767/87/1386-1705$02.00/0

Vol. 138. 1705-1711. No. 6.March 15, 1987 Printed Ln U . S . A .

THEJOURNAL OF i M M U N O L o C Y Copyright (C 1987 by The American Association of Immunoioglsts

ANTIGEN-INDUCED CHANGES IN PHOSPHOLIPID METABOLISM IN ANTIGEN-BINDING B LYMPHOCYTES' CHRISTOPHER D. MYERS,*MARIE K. KRIZ,' TIMOTHY J. SULLIVAN,*'* ELLEN S. VITETTA2*'

AND

From the 'Department of Microbiology, 'the GraduateProgram in Immunology, and 'the Department of Internal Medicine, Southwestern Medical School, Universityof Texas Health Science Center at Dallas, Dallas, TX 75235

Animals. Female BALB/c mice were obtained from Cumberland Farms, (Clinton, TN) and were used a t between 6 and 12wk of age. A number of metabolic changes have been described C3H/HEJ mice were obtained from The Jackson Laboratory (Bar Harbor. ME). in B lymphocytes that occur within a few hours after Preparation of antigen-binding cells (ABCJ.TNP-ABC were puritheir activation with mitogens or anti-immunoglobulin fied as described (22). Briefly, horse erythrocytes (HRBC)(Colorado (anti-Ig). These changes include altered levels of cyclic Serum Co.. Denver, CO) were haptenated with 40 mg trinitrobenzene sulfonic acid (TNBS)/mlof packed erythrocytes. BALB/c splenocytes nucleotides (1-3), Ca++fluxes (4, 5).cytoskeletal changes were prepared by ammonium chloridelysis of erythrocytes and (6, 7). altered serine esterase activity (8, 9),membrane separation of nucleated cells on a single step Ficoll-metrizoate gradepolarization (10, 11). protein kinase C activation (12) dient. Splenocytes and TNP-HRBC were admixed a t a final concenand translocation (13 ) ,decreased membranefluidity (14). tration of 1.5 x lo7 splenocytes/ml and 0.5% packed cell volume TNP-HRBC in balanced salt solution supplemented with 5% fetal and modified phospholipid (PL)3 metabolism (15-21). calf serum (BSS/5%FCS). After mixing, rosettes were formed and Such events precede increases in size, increased expres- were separated by centrifugation on two consecutive Percoll gradients. Rosettes were disrupted by treatment with trypsin and prosion of Ia antigens, and the entry of the cells into cycle. and thelymphocytes were purified ona one-step Ficoll-metriPreviously we (15 ) and others 6) (1 have shown that the nase, zoate gradient. Purified cells were resuspended a t 5 X lo5 per ml of activation of B lymphocytes with a polyclonal antigen RPMI 1640 (M.A. Bioproducts. Baltimore. MD) supplemented with analog, anti-Ig, causes increased incorporation of 32P04 10%FCS (Hyclone, Logan, UT), 50 U/ml penicillin (GIBCO, Grand Island, NY), 50 ml streptomycin (Gibco). 2 mM L-glutamine (Gibco),and 5 x 10-$/M 2-mercaptoethanol(2-ME) (Kodak, Rochester, Received for publication August 8, 1986. NY). Cells were cultured for 18 hr at1 ml per well in 24 well plates Accepted for publication November 14, 1986. (Costar, Cambridge, MA) a t 37°C in a humidified atmosphere conThe costs of publication of this article were defrayed in part by the taining 10%COz. 7% OZ.and 83%Nz. payment of page charges. This article must therefore be hereby marked Antigens. KLH (Calbiochem-Behring. San Diego,CA) and OVA advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- (Sigma Chemical Co., St. Louis. MO) were haptenated by using TNBS cate this fact. as described (23) to give levels of haptenation rangingfrom 18 This work is supported by National Institutes of Health Grants AI21229, AI-19801. and AI-1 1851. MS. Kriz is supported by a grant from molecules TNP per 100,000 Mr KLH (TNPla-KLH) to 57 molecules per 100,000 Mr KLH (TNPs7-KLH)or 10 to 20 molecules TNP per the Welch Foundation. 'Address all correspondence: Department of Microbiology. University molecule of OVA (TNPIo-OVA- TNP,,-OVA). Control reagents derivatized with formyl, mesitylene, and arsonof Texas Health ScienceCenter at Dallas, 5323 Harry HinesBlvd.. Dallas. ate (Ars) were prepared by using formaldehyde sodium bisulfide T X 75235. (Aldrich. Milwaukee. WI). 2 mesitylene sulfonic acid (Aldrich),or p 3Abbreviat10ns used in this paper: ABC. antigen-binding cells; PA. phosphatidic acid; PC, phosphatidyl choline; PE, phosphatidyl ethanol- azophenylarsonate (24). The substitutionratios for formyl and meamine: PI, phosphatidyl inositol; PL, phospholipid: PS, phosphatidyl ser- sitylene derivatives could not be measured spectrophotometrically: ine: RAOVA. rabbit anti-OVA; TD, thymus dependent. however, the chemistry of substitution is the same as with TNBS, 1705


Previousstudieshavedemonstrateda marked into phosphatidicacid (PA)and phosphatidyl inositol (PI), change in the metabolism of phospholipids(PL)after but not into phosphatidyl serine (PS), phosphatidyl ethactivation of resting B lymphocytes with anti-im- anolamine (PE) or phosphatidyl choline (PC).In this remunoglobulin (anti-Ig). In this study we examined port, we purified splenocytes by rosetting to produce a PL metabolisminhighlypurifiedtrinitrophenyl population in which 60 to 80%of the cells bind trinitro(TNP)-bindingB cells after their activation with var-phenyl (TNP)(22). These cells have been used to analyze ious forms of TNP-carrier protein. Such cells show PL changes after stimulation of the cells with thymussimilar changesin PL metabolism when stimulated dependent (TD) antigenssuch as TNP-keyhole limpet with either antigen or anti-Ig,i.e.. increased incor- hemocyanin (TNP-KLH)and TNP-ovalbumin (TNP-OVA). poration of 32P04 into phosphatidic acid and phos- We haveshownthatantigen is capable of inducing phatidyl inositol (PI) but not phosphatidyl choline, changes in PL metabolism similar to those induced by phosphatidyl ethanolamine, or phosphatidylserine. We have demonstrated thatthese responses to an- anti-Ig. The signalinduced by antigen wasdirectly related tigen are TNP-specific and dose-related between 1 both to antigen concentration(1 to 50 pg/ml) and epitope and 50 pglml, producing upto a 2.5-fold stimulation density (TNP,,-KLH to TNP,,-KLH). Furthermore, the sigof 32P04incorporation into PI. The PL response is nal induced by a lightly haptenated carrier, TNPlo-OVA, also directly related to the density of TNP on the was augmented by additional cross-linkingof the carrier carrier and can be augmented by additional cross- with F(ab')2 anti-OVA. These data suggest that altered PL metabolism is a n early antigen-mediated event in B linking of the carrier protein. These data suggest that cross-linking of surface Ig by antigen induces cell activation. a change in PL metabolism as an early event in B MATERIALS AND METHODS cell activation.

1706

ANTIGEN-INDUCED PHOSPHOLIPID METABOLISM CHANGES IN TNP-ABC TABLE I Phospholipid changes in TNP-ABC? 32P04 lncorporation (cprn) Stimulus PI

SI

PA

F(ab’), RAOVA F(ab’), RAMIg TNP.,-KLH TNP3,-KLH

873 f 158 3294 f 595 1.00 1.00 2266 f 285 8109 f 266 2.60 2.46 7643 f 1698 1.69 2.30 1472 f 200 1156f 53 4253f 170 1.32 1.29 “TNP-ABC(3 X 10’) were incubated with 50 pgjml antibody or 35 p g j ml antigen for 45 rninin the presence of 32P04orthophosphate. The reaction was stopped, and the PL were extracted, were separated, and were counted. SI are based on assigning F(ab’], fragments of RAOVA as 1.OO. No changes were seen in labeling of other PL.

mation of plaques (22).It was, however, necessary to test the effects of aspirin on early activation events. In a series of three experiments in whichcells were prepared in thepresence or absenceof aspirin (22).no differences were detected in the PL changes in response to TNP-KLH or F(ab’), fragments ofRAMIg (Table 11).Thus all of the experiments were performed using cells prepared in the absence of aspirin. PL changes induced by dgferent doses of antigen. The PL changes in TNP-ABC exposed to 0.1to 100 &ml TNPw-KLH or TNPlR-KLH were measured a s described. As depicted in Figure 1, thePI changes were dose related in the range of 0.1 to 25 waml and plateaued above 50 wg/ml. The PA response followed a similar pattern when cells were stimulated with TNP5,-KLH; however, the low PA response to TNPle-KLH was difficult to evaluate. Effects of hapten densityof the antigen on P L rnetabolisrn. The results described above indicated that the more heavily haptenated antigen (TNP5,-KLH) was more efficient at eliciting PL changes. This agrees with the notion that cross-linking of surface Ig (sIg) is a n important step in B cell activation. To further test thishypothesis, we prepared aseries of reagentswithdifferent numbers of hapten groups and used them to stimulate TNP-ABC. A s an additional control,TNP5,-KLH was centrifuged at 130,000 X G for 15 min in a Beckman airfuge (Beckman, Palo Alto, CA) to remove aggregates that would increase cross-linking of sIg. A s shown in Figure 2, the changes in PA and PI metabolism of cells stimulated with 50 gg/ml of these reagents for45 and90 min was increased a s the hapten density of the antigen was increased. Removal of aggregates by ultracentrifugation reduced but did not abrogate the stimulation induced by TNP5,-KLH, suggesting that theresponse is not mediated solely by aggregated protein. It should be noted that increased hapten density resulted in a n increased net concentration ofTNP molecules in solution.In Figure 1 the molar concentrations of RESULTS TNP would be approximately equal with100 g g m l TNPlsa r n 1TNPS7-KLH,which produce SI for PI of Response of TNP-ABC to antigen. TNP-ABC respond KLH and 30 w to TD antigen (TNP-KLH) in the presence of carrier- 1.6 and 2.2, respectively. This observation suggests that primed irradiated Tcells by proliferation and differentia- both the hapten density on the carrier and the netcontion (22,30,31).They also proliferate in response toanti- centration of hapten groups inthe cultures influence the Ig and cytokines (32). TableI shows that changes in PL level of the PL changes. This is also supported by the metabolism were detected in TNP-ABC in response to finding that the stimulation by both TNPIR-KLH and F(ab’)2 fragments of RAMIg or to TNP-KLH, within 45 TNPB7-KLHplateaued a t approximately 25 to 50 wgjml. min of stimulation in theabsence of T cells. It was However, the plateau achieved with TNPls-KLHwas sigoriginally shown that thepresence of acetylsalicylic acid nificantly lower than thatachieved with TNP5,-KLH. This (aspirin) duringall rosetting stages of the TNP-ABC pu- suggests that most receptors are saturated at this conrification was necessary to prevent cell activation (32). centration, yet TNP5,-KLH is able to give a “stronger” Using the modified cell purification technique described signal, presumablyby more effective cross-linking of sIg. To examine the effects of cross-linking on PL changes, here, aspirin had no effect on proliferation or the for-


which derivatizes lysine residues. Thus haptenation wasallowed to proceed to completion by using excess hapten to produce a haptena t i m ratio estimated to be equivalent to that of TNP,,-KLH. Conjugation levels of Ars cannot be measuredspectrophotometrically. because lysine, histidine,and tyrosine residues derivatized (each PI are PA of which has a different absorption spectrum). Thus a series of conjugates ranging from “lightly haptenated to ”fully haptenated were prepared. Rernoual of endotoxin. Commercially obtained KLH was tested for endotoxin contamination by using thelimulus lysate assay (Sigma). Such KLH preparations contained 1% w/w endotoxin. To reduce this level of contamination, KLH was dissolved in endotoxin-free phosphate-buffered saline and was recycled extensively over a 5-ml column of ”Detoxi-Gel”(Pierce, Rockford, IL), which reduced the level of endotoxin contamination by 100-fold. KLH purified in this way (“endotoxin-low”)was used to prepare two batches of TNP-haptenated reagent: TNP,,-KLH and TNPZ6-KLH.Lipopolysaccharide (LPS) from S . typhosa 0901for “add b a c k studies was obtained from Difco Laboratories (Detroit, MI: cat. no. 3124-25). Antibodies. Affinity-purified rabbit anti-mouse lg (RAMlg), and rabbit anti-OVA (RAOVA)were prepared and were affinity purified as described (25). F(ab’)n fragments of these antibodies were prepared by pepsin digestion followed by gel filtration (26). PL assays. PL were labeled and were assayed a s described [ 15). Briefly, cells were resuspended in phosphate-free RPMI 1640 with 3%FCS a t 2 to 5 X 1O6/ml. The FCS used was notdialyzed; however, the amount of phosphate added here did not interfere significantly with the uptakeof 32P04.[3ZP04]Orthophosphate (40 pCi] (ICN,Irving, CA) was added to each 200p1 aliquot and was incubated for 30 to 60 min to label PL. Cells were then treated with antigen or a control reagent and were incubated a t 37°C in the continued presence of [ 3 2 P 0 4 ] ~ r t h o p h ~ ~ p hThe a t e .reaction was stopped at various times by the addition of 2 ml cold buffer (15). Thecells were pelleted and were extracted for 3 0 min a t room temperature with a mixture of chloroform, methanol, and 0.04% aqueous CaClz (2:1:0.8) (27, 28). lnsoluble material was pelleted, and the supernatant wasadded to tubes containing 0.5 ml chloroform, 0.5 ml 0.04% aqueous CaC12, and cold carrier lipids. Samples were mixed and were centrifuged, and the aqueous layer was removed. The organic layer was dried under Nz gas, and the lipids were resuspended in 60 pl chloroform and were applied to 20x 20 cm POLYGRAM si1 G plates (Brinkmann. Westbury, NY) that had previously been heated a t 100°C for 1 to 2 hr. Sampleswere dried onto the plates with a stream of air andwere chromatographed in two dimensions (29). The firstdimension consisted of chloroform, methanol, ammonium hydroxide, and water [65:35:2.5:2.5). and the second consisted of chloroform, acetone, ethanol, acetic acid, and water (50:22:9:10:5).Plates were dried for 1 5 min between developments. Lipids were stained with l2 vapor. were compared with co-chromatographed purified standards, and spots co-migrating with cold PA, PC, PS, PE, and PI were identified, were cut from plates. and were counted. In manyexperiments, affinity-purified F(ab’)z fragments of RAOVA were useda s a control. In six experiments comparing this reagent with medium alone, the stimulation indices (SI) were 1.02 k 0.56 for PA labeling and 1.10 f 0.11 for PI. Cell proliJeration. Cell proliferation was assayed as described (1 5). Briefly, cells were resuspended in complete medium a t 5 x 105/ml, and 100p1 were added to eachwell of a 96 well microtiter plate. Test reagents in complete medium were added a t a final vol of 200 al. Cells were cultured as described above for 2 or 3 days and were pulsed with 1 pCi per well [3H]thymidine. After a n additional 18 hr. culture cells were harvested, andthymidine uptake was assessed by scintillation counting.

1707

ANTIGEN-INDUCED PHOSPHOLIPID METABOLISM CHANGES IN TNP-ABC TABLE I1 Effect of including asplrln during the rosetting stages of the TNP-ABCpurfficatlon" SI (+ SD) Stimulus

PA

+ Aspirin 1 .oo

Flab'), RAOVA F(ab'), RAMlg 2.29TNP57-KLH TNP3,-KLH

3.89(+ 0.68) 2.01 1.44 1.45 (+ 0.37) 1.51

PI - Aspirln

1 .oo 3.71 (+ 0.53)4.02 1.42 (+ 0.31) 1.96

+ Aspirin 1 .oo

- Aspirin 1 .oo

(k0.47)4.28

(k0.46)

(k0.57) 2.09

(k0.30)

TNP-ABC were prepared as described either withor without 0.4m d m l aspirin during all stages in whichcells B were in contact with hapten (22).Aspirin was removed before overnight culture and was not added to any experimental of 32P04 mixtures. TNP-ABC (2.5x lo5)were incubated with 50 pdml of the various stimulators in the presence orthophosphate before extraction and analysisof PL. Cells treated with F(ab'), RAOVA were given a value of 1 .OO. The data represent the meank SD of three experiments except for TNPS7-KLH.which was used in only one experiment. a

westimulatedcellswith TNPIo-OVA and then crosslinked the cell-bound antigen with the same F(ab'I2 fragx ments of RAOVA normally used as a negative control.A s < 2 H shown in Figure3, TNPlo-OVAalone gave essentially no ._ 6 stimulation of PA or PI metabolism. When TNPlo-OVA 1.5 was cross-linked with RAOVA, PL metabolism was sigE'._ ci nificantly increased. Specificity controls. A number of approaches have 1 been used to show that changes in PL are mediated by J 0 25 50 75 100 0 5025 75 100 the antigen-specific receptors on the B cells. To investiAntigen Concentration (pg/ml) gate this issue, we first attempted to block the antigeninduced changes in PL with monovalent hapten (TNPFigure 1 . The dose response of 3zP04 incorporation intoPA (0.W) and PI (0.0)after stimulationof TNP-ABC with TNP-KLH. TNP-ABC (2X lo5) Lys). Initial examination of the data suggested that the were incubated with various concentrations of TNPm-KLH (Panel A] or blocking wasvery efficient.However, it became apparent TNPls-KLH(Panel B)for 90 rnin in the presenceof =PO4 orthophosphate. that this was due to the presenceof inorganic phosphate Control cells were treated with 50 pglml F(ab'), fragments ofRAOVA. The SI is the ratio of incorporation into test cells compared with control in thecommercial preparation of TNP-Lys (ICN Biomedicells, The data represent the mean (+SD] from three similar experiments. cals, Irvine,CA), which waslowering 32P0, incorporation by dilution. Repeated attempts to remove the phosphate by chromatography on Sephadex G- 10 or recrystallizaA. B. tion from hot aqueous solution did not generatea reagent 2 that was sufficiently free of phosphate to use in these experiments. 1.75 The second approach designed to demonstrate the an2.5

f


4

TT

1.50

X

RAMIg

1.25

a

U

=

2.25

&

j

i

TNPqo-OVA

I

= 0

TNPlo- OVA +RAOVA

2

RAOVA

175

I

I

I

I

I

2

3

4

1.50

RAMIg

1.25

TNPqo-OVA

1

TNPIO- OVA + RAOVA

3

8

3

8

Figure 2. The effect of hapten density on changes in PL metabolism. TNP-ABC (2x lo5]were stimulat6d with 50 pglml TNP-KLH prepared at various hapten densities. Spun TNP,,-KLH represents TNPs7-KLH that was airfuged for15 min immediately before use to remove aggregates. S I were compared with those of untreated cellsor to cells treated with F(ab'), fragments of RAOVA and were assayed45 (PanelsA and B)or 90 (Panels C and D ) min after incubation of the cells. Panels A and C represent incorporation into PA, and PanelsB and D represent PI. The data represent the mean(kSD)from three similar experiments.

RAOVA

I t I

1

1

I

2

3

4

STIMULATION I N D E X Figure 3. The effectof secondary cross-linkingof antigen on changes in PL. TNP-ABC(2X lo5)were incubated with F(ab'], fragments of RAMIg at 50 p d m l or TNP,,-OVA at 35 pglml in the presence of 32P0, orthophosphate. Fifteen minutes later. F(ab')2 fragments of RAOVA were added to 150 p d m l in some TNP,,-OVA samples. All samples were incubated for another 90 min before PL were assayed. (W) PA: (Ed) PI. The data (+SD) from six similar experiments. represent the mean responses

1708

ANTIGEN-INDUCED PHOSPHOLIPID METABOLISM CHANGES IN TNP-ABC


TABLE 111 tigen-specific nature of the PL changes was to use proEffects of uarlous soluble antlgens on phosphollpid rnetabollsm of teins derivatized with other haptens. The methodsused normal B cells" to add formyl and mesitylene groups, a s with TNBS, SI [iSD) derivatizes only lysine residues. Thesehaptens also show Stimulus PA PI structural similarities to TNP and would be expected to 1 .oo F(ab'), RAOVA 1 .oo havesimilareffects on the overall protein structure. 3.89(+ 1.51) 3.81 (+ 0.36) F(ab'), RAMlg However, these derivatizations do not give rise to a col0.95(& 0.30) 1.13 (+ 0.30) TNPJI-KLH 1.04(+ 0.17) 1.28(?0.11) TNPsg-KLH ored product; thus the level of haptenation cannot be 0.90(C0.06) 1.14(+ 0.07) TNP33-KLH measured spectrophotometrically. Therefore haptenation 0.91(+ 0.1 1) TNPla-KLH 1.15(+ 0.07) with these reagentswas performed under conditions simMesitylene-KLH 0.88 (+ 0.16) 1.04(+ 0.02) (+ 0.15) 0.92 Fomyl-KLH 1.04(k 0.02) ilar to those used for TNP,,-KLH, i.e., excess reagentand 0.78(+ 0.29) ArSH-KLH 0.91(+ 0.25) overnight incubation at room temperature. We assume 0.84 I+0.19) 0.98(+ 0.081 Ars,-KLH that the reaction was as close to completion a s with "T cell-depleted spleen cells (2.5X lo5)were incubated with50 &/ml TNBS, producing a similar level of substitution. of various reagents for90 mln in the presence of 32P04 orthophosphate of PL. Cells stimulated with F(ab'), RAOVA Haptenation withArs produces a colored product. How- before extraction and analysis SI of 1 .OO.The data represent the mean (+ SD) of were assigned a control ever, because three amino acid residues are substituted, three experiments. each producing a different absorbance spectrum, accurate determinations of the level of haptenation were not TABLE 1V possible. Instead, two of the seriesof reagents produced; Mitogenic effects of haptenated KLH" the two most heavily haptenated (designated ArsH-KLH SI and Ars,-KLH) were judged to be closest to TNPs7-KLH Normal B TNP-ABC and were used to investigate PL changes. As shown in cells Figure 4, although PL changesin TNP-ABC occurred 1 .o Medium 1 .o NDb LPS 43.1 when cells were cultured withTNP-KLH,no changes were 14.4 TNPs7-KLH 16.8 observed with the use of mesitylene-KLH, formyl-KLH, TNP8,-KLH 6.8 15.9 or Ars-KLH. The final test for specificity was to demon7.5 TNPss-KLH 23.4 5.5 13.6 TNPXS-KLH strate thatnone of the haptenated reagents would induce ND 19.8 ARSH-KLH PL changes in a population of normal B cells. Table 111 ND 7.5 ARSw-KLH shows that changesin PL metabolism did not occur in T ND 39.7 Formyl-KLH Mesityl-KLH ND 42.3 cell-depleted spleen cells cultured withany of the hapten4.1 RAMIg ( 100 pg/ml) ND ated proteins. 3.1 RAMIF! (50uclmll ND Functional testing of antigens. Previous studies (30, a TNP-ABC or T cell-depleted splenocytes from BALB/c mice were cul3 1) have shownthat neither TNP-ABC nor normal B cells tured at 2 x lo5 cells per 200 pl with 50 pg/ml of various test reagents. pulsed with l pCi [3H]thymidineand proliferate in response to TNP-KLH in the absence of T After 48 hr of incubation, cells were were incubated fora n additional 18 hr before harvesting. j3H]Thymidine cells. TNP-ABC, but not normal B cells, will respond to incorporation was assessedby scintillation counting. The data represent were obtained in a TNP-KLH when carrier-primed T cells are added. F(ab')2 the mean of triplicate determinations. Similar results second experiment. In additional experiments, TNP-OVA and RAOVA anti-Ig will produce a low levelof thymidine incorporation gave no detectable mitogenic effects (data not shown]. in normal B cells when added at high concentrations (33). ND. not done. but gives a substantial response when low concentrations are added in the presence of IL-4 (BSF-1)(34, 35). To confirm the functional activities of our reagents, we first attempted to demonstrate that they produced no Formyl Mesitylene TNP Ars proliferation of the TNP-ABC in the absence of T cells OH , and cytokines. As can be seen in Table IV, this was not HO-ASSO OH T T the case. F(ab'), fragmentsof RAMIg at 50 and 100 pg/ ml produced the expected small burst of early proliferation. However, unexpectedly, the TNP-KLH preparations tested produced SI of 5.46 to 14.4 in the proliferation assay. Additional experiments showed this response to be dose related, with a detectable response to TNP,,-KLH and TNP,,-KLH at 1 pglml and 10 pg/ml, respectively (data not shown). Because this response was unexpected, even though the antigen concentrations were much higher than previously reported, we examined the antigen specificity of this response by testing all of the haptenated reagents on normal B cells. Table IV shows that this stimulation is polyclonal, occurring in response to all forms of haptenated KLH. Subsequently,limuluslysate assays revealed that thecommercial preparations of KLH used for analogs. TNPspecific antigen and antigen Figure 4. PL responses to the haptenations contained 1%endotoxin, and the conABC (2 x lo5)were treated with various reagents at 50 pg/ml in the presence of 32P04 orthophosphate for 90 min before assaying the incor- tamination of all the haptenatedKLH reagents used was poration of 32Pinto PL. The control value represents cells stimulated withsimilar. This finding was supported by the fact that the 50 puml F(ab'Iz fragmentsof RAOVA. (B)PI; (W) PA. The data represent proliferative response to TNPS7-KLH waspartially inthe mean (?SD) of three experiments.


1709


TABLE V hibitable by polymyxin B (data not shown). Effects of LPS levels on phospholipid metabolism’ A s shown previously, LPS does not inducePL changes SI (& SD) in normalB cells and does not affect the anti-Ig-mediated Stimulus PA PI changes in PL metabolism (16). To confirm that our 1 .oo 1 .oo previous data were due to stimulation with specific anti- F(ab’), RAOVA 3.63 (k 0.23) 3.89 (f0.35) gen and not LPS, werepeated the PL studies with the use F(ab’)2RAMIg Anti-Ig + 50 pdml LPS 3.55 (20.04) 3.84 (f 0.46) of TNP-ABC purified from the LPS low responder mouse LPS 150 u d m l l 0.96 I f 0.031 0.92 Ik 0.011 1.14 if o . l l j 0.99 ik 0.06j strain, C3H/HEJ. Figure 5 shows thatthesame PL LPSi5 p z m l ) ’ 1.30 (f0.37) 1.01 (k0.04) changes occurred in these cells as those observed by KLH Endotoxin-law -KLH 1.21 (k 0.25) 1.03 (f0.15) using TNP-ABC from BALB/c mice. Furthermore, LPS TNP5-I-KLH 2.00 (% 0.37) 2.69 (f 0.55) 1.98 (% 0.43) 2.30 (f 0.37) had no effect on the PL metabolism of these cells, and Endotoxin-low TNPsI-KLH TNPS1-KLH+ 2.15 (k 0.24) 2.32 (k0.42) the addition of LPS to TNPS7-KLHor TNP,,-KLH did not Endotoxin-law LPS (5 pglml) increase the responses to these antigens. Also, we found TNPIcKLH 1.40 (+ 0.23) 1.79 (k 0.27) 1.32 (k 0.18) 1.59 (k0.18) a 50-fold lower sensitivity of C3H/HEJ B cells to S . ty- Endotoxin-low TNPZ6-KLH Endotoxin-low TNPZ6-KLH+ 1.37 (k 0.14) 1.59 (k 0.18) phosa LPS but little difference in the sensitivity to mitoLPS (5 pglml) genesis by TNP-KLH as compared with BALB/c B cells. “TNP-ABC (2.5 X lo5) were incubatedwith 50 pg/mlof the listed To examine the role of endotoxin in thePL response of reagents for 90 min in thepresence of 32P04orthophosphate before TNP-ABC, a series of reagents was produced with de- extraction and analysisof PL. Cells stimulated with Flab’), fragments of RAOVA were assigned a control S I of 1.00. The data represent the mean creased levels of LPS contamination as measured by the (+ SD) of four separate experiments. limulus lysate assay. Extensive recycling of KLH over a n LPS affinity matrix(Detoxi-Gel)resulted in a99%reducDISCUSSION tion in the level of endotoxin contamination to 0.1 pg LPS/mg of KLH. With the use of this KLH, derivatives The data presented in this report confirm and extend haptenated at 51 and 26TNP molecules per 100.000 Mr previous studies from our own laboratory (15)and others KLH were produced. When tested in thymidine uptake (16-2 1)concerning changesin PL metabolism during the assays on normal B cells, the KLH was 10-fold less mi- early stagesof B cell activation. Thus antigen,similar to togenic. However, the haptenated derivatives still pro- anti-Ig induces increases in PI and PA metabolism. Analduced a n increase in the incorporation of thymidine, but ysis ofPL changes have been carried out in TNP-ABC, this response was less sensitive to inhibition by polywhich contain 60 to 80% TNP-binding cells (22). The myxin B, suggesting that either the haptenated protein response of such cells to anti-Ig stimulation was a 3.54may be mitogenic itself or it may be contaminated with fold (f0.63;n = 33)increase in incorporation of inorganic some other mitogen. phosphate into PA and a 3.91-fold (f0.45: n = 33) inTable V shows theeffect of these purified reagents on crease in incorporation into PI. Treatment of the same the PL metabolism ofTNP-ABC. As can be seen, no cells with TNP-KLH caused a similar pattern, i.e., indifferencesin theresponses were observed by using creased incorporation into PA and PI. However, the level either the purified or non-purified reagents; nor did the of stimulation was always muchlower with antigen, and re-addition of excess LPS to the purified reagents result there wasa more significant differencebetween the stimin any changesin the responses. Furthermore, inagree- ulation indices for PA and PI. ment with published data (16). the addition of very high There are a number of possible explanations for the concentrations of LPS (50 &/ml) to cells stimulated with different magnitudes of the response, the most likely anti-Ig resulted in no change in PL metabolism (nor did being that antigen activatesa smaller percentage of cells added LPS alterthe dose responsecurve of purified than does anti-Ig, or that anti-Ig provides a “stronger” TNP,,-KLH; data not shown). cross-linking signal than antigen by binding to many epitopes on the Ig receptor. Although the first alternative is undoubtedly responsible for some of the differences observed, the larger difference between PA and PI stimulation, especially at low concentrations of lightly haptenated protein, and the ability of secondary cross-linking to increase the response adds support to the second hypothesis. Another important question raised by these studies is why such high concentrations of antigen or anti-Ig are required to produce measurable PL responses a s compared with those required to produce proliferation. It is known that PA and PI are intermediates in a cycle ofPL metabolism (36),which may lead to the production of the inositol phosphates and diacylglycerol (20, 36). These molecules could be active in the induction of Ca++ fluxes and protein kinase C activation (20, 37). ThusPA and PI are only intermediates in a cycle, being rapidly synthesized and degraded. In this case, a very small change in Figure 5. Changes in PL metabolism inTNP-ABCfrom C3H/HEJ mice. TNP-ABC (2 x lo5)were treated with various reagents at50 pglml in the turnover may be required to produce a maximum biologpresence of 32P04 orthophosphate. After 90 min, the PL were analyzed. ical effect, andto see a responsein our assay system, the The data represent the mean responses from duplicate determinations. PA (m); PI (EA). Similar results were obtained in a second experiment. cells require “hyperactivation.” It must be emphasized

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Acknowledgments. We thank Ms. T. Wilson and Ms.

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25. Pure. E., and E. S.Vitetta. 1980. The murine B cell response to TNP-


that the present assaysystem is a dynamic system mea- L. Trahan for expert technical assistance and Ms. G. A. suring levels of label incorporated intothe intermediates Cheek, Ms. Cindy Baselski, and Ms. F. Hallfor the careful at a particular time. No pharmacologic reagents have preparation of the manuscript.We are especially grateful been added to inducea build up of the labeled compounds, to Drs. E. C . Snow, S . A. Grupp and J. A. Harmony for although there may be considerable build up of label in helpful discussions of unpublished data andmethods. the large pools of cellular PA and PI. REFERENCES Previous studies suggest that changes in PL metabolism are the signal transducing mechanismfor cell acti1 . Kaever. V., and K. Resch. 1985. Are cyclic nucleotides Involved in the initiation of mitogenic activation of human lymphocytes. vation by antigen or anti-Ig (15-17, 20) but not LPS (16, Biochim. Biophys. Acta 846:216. 38). Treatment of B cells with intact anti-Ig results in Watson, J. 1976. The involvement of cyclic nucleotide metabolism 2. Ca++fluxes, but much smaller changes PL in metabolism in theinitiation of lymphocyte proliferationinduced by mitogens. J. Immunol. 1 17:1656. than induced by F(ab’), anti-Ig (37).and suchcells do not 3. Largen, M. T..and B. Votta. 1983. immunocytochemical evidence progress through cycle a s they do when stimulated with for 3’.5’-cGMP and 3’.5’-cGMP-dependent protein kinase involvement in lymphocyte proliferation. J . Cyc. Nuc. Prot. Phosph. Res. F(ab’), anti-Ig. This suggests that the PL changes are a 9:23 I . consequence of activation by anti-Ig. The present data 4. Braun, J.. R. I. Sha’afi, and E. R. Unanue. 1979. Crosslinking by support the hypothesis that cross-linking of surface religand to surface Immunoglobulin triggers mobilization of intracellular 45Ca2+in B lymphocytes. J . Cell. Biol. 82:755. ceptors results in changes in PL metabolism, and these 5. Ransom, J. T.,D. L. Diguisto, and J. C. Cambier. 1986. Single cell changes are unaffectedby the presence of high concenanalysis of calcium mobilization in anti-immunoglobulin-stimulated trations of LPS. B lymphocytes. J . Immunol. 136:54. 6. Schreiner, G . F., K. Fujiwara,T. D. Pollard,and E. R. Unanue. We have demonstrated that the changes in PL metab1977. Redistribution of myosin accompanying cappingof surface Ig. olism are both hapten specific and dose related in the J . Exp. Med. 145:1393. presence of contaminating LPS. Thus thepossible effects 7. Rogers, K. A., M. A. Khoshbaf. andD. L. Brown. 198 1 . Relationship of microtubule organization in lymphocytes to the capping of lmofLPS have been studied. Commercially obtained KLH munoglobulin. Eur. J . Cell. Biol. 24: I . was used in the initial experiments. This KLH contains 8. Kishimoto, T., H. Kikutani, Y. Nishizawa. N. Sakaguchi, and Y. Yamamura. 1979. Involvement of anti-lg-activated serine protease 10 pg LPS per mg KLH. Extensive recycling of the KLH In the generationof cytoplasmic factor(s]that areresponsible for the over the “Detoxi-Gel”affinity matrix to remove LPS retransmission of Ig-receptor-mediated signals. J . Immunol. 123:1504. sulted in lowering this contamination to approximately 9. Kishi. H., Y. Miki, H. Kikutani. Y. Yamamura. and T. Kishimoto. 1983. Sequential induction of phospholipid methylation and serine 0.1 pg LPS per mg KLH. Although this abrogated most of esterase activation in a B cell differentiation factor (BCDF)-stlmuthe mitogenicity of native KLH, haptenated derivatives of lated human B cell line. J . fmmunol. 131:1961. this purified KLH maintained significant polyclonal mi- 10. Monroe, J. G., and J. C. Cambier. 1983. B cell activation. 1. Antiimmunoglobulin-induced receptor crosslinking results in a decrease togenic activity that was only partially inhibitable by of murine B lymphocytes. J . Exp. in the plasma membrane potential polymyxin B. This mitogenic effect was also noted in Med. 157:2073. C3H/HEJ cells that were 50-fold less responsive to S. 1 1 . Cambier, J. C., and J. G . Monroe. 1984. B cell activation. V. Differentiation signalling of B cell membrane depolarization, increased Ityphosa LPS. Thus it is possible that haptenated KLH A expression, Go to G I transition, and thymidine uptakeby anti-lgM shows some nonspecific mitogenicity. Alternatively, it is and anti-IgD antibodies. J . Immunol. 133:576. possible that some other molecule(s) in this preparation 12. Monroe, J. G., J. E. Niedel, and J. C. Cambier. 1984. B cell activation. IV. Induction of cell membrane depolarization and hyper-I-A is responsible for the mitogenic effect. expression by phorbol diesters suggests a role for protein kinase C in murine B lymphocyte activation,J . Immunol. 132:472. Given these considerations, the data suggest that the 2.2..K. M. Coazeshall. andJ. C. Cambier. 1986. Transloca. . changes in PL metabolism are independent of changes 13. Chen, tion of protein kinaseFduringmembrane immunoglobulin-mediated mediated by endotoxin. The response is antigen specific transmembrane signalling in B lymphocytes. J . Immunol. 136:2300. and is not induced by any dose of LPS. Secondly, TNP- 1 4. Keating, K. M.. D. A. Roess, J. S. Peacock, andB. G . Barisas. 1985. Glucocorticoid effects on membrane lipid mobility during differentiaABC purified from C3H-HEJ mice (an “LPS-low retion of murine B lymphocytes. Biochim. Biophys. Acta846:305. sponder”) showed no difference in PL responses. Fur1 5. Kriz, M. K., E. S. Vitetta. and T. J. Sullivan. 1986. Changes in phospholipid metabolism during B lymphocyte activation. J . Immuthermore, reduction of LPS levels by 99% or addition of nol. 137:478. LPS at higher concentrations had no effect on the PL 1 6. Grupp, S.A.. and J. A. K. Harmony. 1985. increased phosphatidylinositol metabolismis a n important but nota n obligatory early event response either to haptenated proteins or to anti-Ig. The B IvmDhocvte activation. J . Immunol. 134:4087. possibility remains that a very low concentration of LPS 1 7. in Kenny, b. J.,