Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
This information is current as of August 3, 2017. Pillars Article: Identification of a Macrophage Antigen-Processing Event Required for IRegion-Restricted Antigen Presentation to T Lymphocytes. J. Immunol. 1981. 127: 1869− 1875. Kirk Ziegler and Emil R. Unanue J Immunol 2007; 179:5-11; ; http://www.jimmunol.org/content/179/1/5.citation Permissions Email Alerts Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 Subscription 0022-1767/81/1275-1869$02.00/0 THE JOURNAL OF hMUNOLOGY Copyright @ 1981 by The American Assoclation of ImmunOlOgiStS VOI.127, NO 5. November 1981 Pnnted In U.S A IDENTIFICATION OF A MACROPHAGE ANTIGEN-PROCESSING EVENT REQUIRED FOR REGION-RESTRICTED ANTIGEN PRESENTATION TO T LYMPHOCYTES' KIRK ZIEGLER AND I- EMlL R. UNANUE From the Departmentof Pathology, Harvard Medical School, Boston,MA 02 1 7 5 phosphate-buffered saline (PES) containing 1 mCiof carrier-free Na'251 (New England Nuclear, Boston, MA) and 50 pg of chloramine-T (Eastman Kodak, Rochester, NY) were incubated for 1 0 min at 4°C. Sodium metabiMacrophages are intimately involved in the antigen-specific sulfite (Sigma Chemical Company, St. Louis, MO) (20 pI of a 5 mg/ml activation of L y l +2-3- T lymphocytes (reviewed in 1-3). The solution) was added to terminate the reaction, and then the labeled bacteria initial event in T cell activation is thought to be the recognition were washed 5 times in PBS to remove free '251, Bacteria thus treated of the immunogen and I region gene products present on the showed 1 to 2 counts per minute (cpm) per bacterium. More than 99% of the radioactivity was precipitable in1090trichloroacetic acid(TCA). Listeria macrophage cell surface (3, 4). In the present study, we monocytogenes labeled with lZ5l ('251-Listeria)were stored at 4°C in PBS attempt to correlate the uptake, ingestion, and catabolism of for use over several weeks and were washed immediately before use. In 1 experiment, Listeria monocytogenes were attached to culture dishes antigen by the macrophages to their ability to present immuusing poly-L-lysine. Tissue culture wells (1 6 mm diameter) were treated (1 nogeneic molecules to T cells. To this effect, we have used the hr, 20°C) with an aqueous solution of poly-L-lysine (1 mg/ml) and then binding of specific T cells to macrophages as a functional washed. Heat-killed Listeria inPES (0.5 ml of IO' bacteria/ml) was added, assay of macrophage-associated antigens. It is known that the the plate was centrifuged (800 X G, 5 min), and then incubated for 1 hr at interactions between T cells and macrophages bearing antigen 20°C. After washing with PES. a confluentlawn of bacteria remained firmly attached to the dish. may be analyzed directly by quantitating the antigen-specific Uptake and ingestion of Listeria by macrophages. Iodinated Listeria physical interactions taking place between both cells (5-1 3). monocytogenes was added to 3 X l o 5 to 1O6 peritoneal exudate cells This quantitation of macrophage-associated antigenicity within (PEC)' planted in 16-mm diameter tissue culture wells (Falcon No. 3008). The plates were centrifuged (800 x G, 5 min) and then incubated as a short temporal framework is a unique way to study the required in the differentexperiments. The macrophages were then washed mechanisms of macrophage handling of antigen in the context thoroughly, and Triton X-I00 to a 1 .O% concentration was added. Radioof I region gene effects. Previous studies have approached this active counts were made on the solubilized macrophage extract. The ingestion of Listeria was followed microscopically.Heat-killed Listerla (0.3 ml of 5 X 106/ml) were added to macrophage monolayers formed on 15-mm diameter coverslips resting ontissue culture wells. The plates were Received for publication April 29, 1981. centrifuged (800x G.5 min) and then washed to remove unbound bacteria. Accepted for publication July 28, 1981. The macrophages were then incubated for various periods of time (0to 120 The Costs of publication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked advertisement in min) at 37°C before fixation with 190 paraformaldehyde. The Listeria monaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by National Institutes of Health GrantsCA 14723, Abbreviations usedin this paper: HEPES,N-2-hydroxyethylp1perazine-N"2AI 14732, and A I 17259. ethanesulfonic acid; PEC. perltoneal exudate cells. 1869 Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 problem by using radioactively labeled antigen molecules to The mechanism of macrophage-antigen handling was follow their fate in phagocytic cells, and long-term functional studied using a system that involves the quantitation of assays for the immunogenicity of macrophage-associated anthe antigen-specific binding of Listeria monocytogenesimmune T cells to macrophages. Specific T cells did not tigen (1 4-1 6). The phagosome-lysosome pathway of antigen bind to native antigen. Because the specific binding of T uptake and degradation and the potent immunogenicity of cells to macrophages could be measured during a short macrophage-associated antigen have been well illustrated. (5- to 18min) interaction, it was possible to follow the However, because of the dynamic nature of macrophage-antitemporal development of a T cell-binding substrate with gen handling events and the length of time required to funcincreasing time ofantigen-macrophage interaction.In tionally assess antigenicity, precise cause-and-effect relationcontrast to the rapid (5-min) uptake of Listeria by macships between antigen-handling events and immune recognirophages, the developmentofTcell-bindingabilityretion could not be determined with certainty. quireda 3 0 to 60min period ofantigen-macrophage Wehave employed as antigen the intracellular pathogenic interaction. During this processing period, Listeria orgabacteria Listeria monocytogenes. The activation of Listerianisms bound to the macrophage surface were ingested immune T cells requires macrophages in a process regulated and partially catabolized. Unlike antigen uptake, antigen by the I region of the H-2 (1 1 , 12, 17). Moreover, Listeria can processingwasatemperature-dependentandenergyrequiring event. Although macrophages treated with be traced in the macrophage and its uptake, ingestion, and paraformaldehyde before antigen processing did not de- catabolism monitored reasonably well. We now define a macvelop T cell-binding activity, macrophages treated with rophage processing event required for the T cell recognition of paraformaldehydeafter a W m i n antigen-processing pe- Listeria antigen. riod retained T cell-binding ability. The kinetics of antigen MATERIALS AND METHODS catabolism correlated with antigen processing, and inhiMice. A/St mice were purchased from West Seneca Laboratories, Bufbition of antigen catabolism was associated with a corfalo, NY. Male or female mice at 8 to 12 wk of age were employed. respondinginhibitionofantigenprocessingforTcell Listeria monocytogenes. The preparation of Listeria monocytogenes was binding. Anti-la antibodies had no effect on Listeria up- previously described (1 7). Live bacteria were used to immunize mice, and take or catabolism. These results supply direct evidence heat-killed organisms served as antigen for use in vitro. for a macrophage-antigen processing event relevant to T Heat-killed Listeria monocytogenes were labeled with '251 by the chloramine-T method (1 8). Briefly, 10' washed bacteria suspended in 130 pl of cell recognitionof antigen. 1870 KIRK ZIEGLER AND EMlL % specific binding = x- Y X 100 X Anti-Listeria T cells were measured by 3 assays described in full detail in previous studies (1 1, 12). These were: 1) the production of thymocyte mitogenic protein (17);2) the development of tumoricidal activity by macrophages; and 3) T cell proliferation. All 3 assays require an interaction between immune T cells and macrophages modulated by the I region ofthe H-2 (1 7, 19). In the assay for thymocyte mitogenic protein, T cells and macrophages were co-cultured for 24 hr, and then culture medium was tested for mitogenic activity on A/St thymocytes. In the cytocidal assays, after 24 hr of cultureof macrophages and T cells, the T cells were removed, and "Cr-labeled P-815 mastocytoma cells were added to the macrophages for a period of 18 hr. Percent specific cytolysis was determined from the release of 5'Cr by the tumor cells (11, 19).T cell proliferation wasassayed after 4 days of culture of T cells with macrophages. Antibody reagents. The monoclonal antibody anti-LAh represents the culture supernatant from a hybridoma cell line (clone 10-2.16) originating inthelaboratory of Dr. L. A. Herzenberg (20). A.TH-anti-A.TL(anti-la') antiserum was purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). The strength and specificity of these reagents were determined by standard serologic means employing complement-mediated lysis of appropriate spleen target cells. UNANUE [VOL. 127 immunofluorescence. The assessment of macrophage cell surface la molecules was performed by indirect immunofluorescence with the monoclonal anti-LAh (21 ). RESULTS The binding assay. The antigen specificity and macrophage dependence of antigen recognition by Listeria-immune T cells is illustrated by the experiment of Table 1. Listeria monocytogenes-immune T cells were added to tissue culture dishes containing the various binding substrates. Binding was monitored by testing the nonadherent T cells in the macrophage cytotoxicity assay. Although complete depletion of Listeriaspecific T cell activity was observed with macrophages incubated with Listeria, no binding was observed with macrophages incubated with the antigenically unrelated bacteria, Safmoneila typhi. Furthermore, Listeria-immune T cellsdid not bind to Listeria monocytogenes attached to the dish in the absence of macrophages. The inability of immobilized Listeria monocytogenes to support binding was also observed when the mitogenic protein and proliferation assays were used to assess T cell activity (data not shown). Clearly, both antigen and macrophages are required to generate a substrate to which T cells will bind. Kinetics of antigen handling. Several aspects of the handling of Listeria by macrophages were studied. Macrophages from Listeria monocytogenes-immune mice containing a high proportion of la-positive cells were used (21). Such macrophages have been shown to bind Listeria-immune T cells much better than macrophages from peptone-induced PEC (unpublished observations). The uptake, ingestion, and catabolism of Listeria by macrophages as function of time are shown in Figure 1. Substantial uptake of Listeria occurred within 5 to 10 min by using a centrifugation step (800 X G, 5 m i d to initiate the interactions of bacteria with macrophage monolayers (Fig. 1a). Routinely, 15 to 30% of the added '251-Listeria became macrophage associated during this time period. Rapid (5-minute) uptake occurred equally well in the presence of both sodium azide (10" M) and 2-deoxyglucose (10" MI, and also at 4OC (data not shown). After the rapid binding (5 min) of Listeria to the macrophage cell surface, ingestion was evidenced by the progressive decrease in the number of macrophage surface-associated bacteria, reaching a 50% reduction by about 10 min (Fig. 16). In Figure I C , macrophages were exposed (30 min 37OC) to '251-Listeria to allow for uptake and ingestion, then washed thoroughly; the radioactivity in the macrophage and the superTABLE I AnOgen recognition by Listerla-immune T cells: specifioty and macrophage dependence" Activity of Nonadherent Binding Substrate T Cells: MacrophageMediated Cytolysis (% i SEM) ?4 Spectfic Binding ~ Macrophages Macrophages and Satmoneffa Macrophages andListeria 39.5 f 3.9 43.9 f 1.9 -5.4 k 1.6 Culture dish Salmonella Listeria 42.7 k 0.9 47.3 i 0.6 50.3 k 5.7 "7 1 100 -1 1 -18 Macrophage monolayers (1.5 X lo6 PEC per well) were incubated (1 hr. 37°C) with heat-killed Salmonella typhi or Listeria monocytogenes(0.3 pl of IO' bacteria per ml). Some culture dishes contained bacteria attached to the culture surface. Listeria-immune T cells (5 X lo6 T cells per well) were added to the dishes containing the indicated binding substrate: the plates were centrifuged (50 x G. 5 min. 20°C) andthenincubatedat 37OC for 1 hr. The T cells for Specific ionadher& to tile binding substrates were recovered and tested activity in the macrophagecytotoxicity assay. The activityof lo5cells per well is zk SEMof duplicatebinding givenasthemeanpercentspecificcytolysis reactions. Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 ocytogenes organisms on the surface of the macrophages remained reactive with antidistena antibody and could bevisualized by indirect immunofluorescence. Inverted coverslips were incubated (30min, 4°C) on 20 pl of a 1:IO dilution of rabbit anti-Listeria serum, washed, and then treated with fluorescein-conjugated F(ab% goatanti-rabbit globulin (20 pl of 50 pg/ml). Catabolism of '%Lisferia b y macrophages. Macrophage monolayers were cultured with '251-Listeriato allow uptake (as described above) and then washed thoroughly. After incubation for various periods of time, the supernatant was removed, and Triton X-1 00 (1 % v/v in H20)was added to the macrophages. The solubilized macrophages were recovered from the dish with the aid of a rubber policeman. Both the macrophage andsupernatant fractionswere incubated with anequal volume of 20% TCA. and the precipitate was separated by centrifugation (1000 x G. 30 min, 4°C). Radioactivity was measured in the precipitate and in the soluble supernatant. 7 c e h andmacrophages. The preparation of cell populationswas exactly as described previously (1 1, 17, 19).Briefly. T cells were purified from the peritoneal exudates of mice infected i.p. with 1 to 5 x lo4 live Listeria organisms. After 1 wk, the mice received an i.p. injection of 10% proteose peptone and then were sacrificed 3 days later. T cell enrichment, accomplished by the removal of cells adherent to tissue culture dishes and nylon wool, routinely resulted in a more than 95% Thyl.2-positive lymphocyte population containing lessthan 0.5% macrophages. Normal and Listeria monocytogenes-immune mice (as described above for T cells) were used as macrophage sources. In both cases, PEC were harvested 3 days after an i.p. injection of proteose peptone(1 0% w/v). The PEC were incubated (2 hr at 37°C) in tissue culture vessels to allow macrophage adherence, and then the nonadherent cells were removed by washing. Media. Peritoneal lavage was performed wtth Hanks' balanced salt solution containing 2% fetal calf serum, 10 mM HEPES' buffer, and heparin ( I O U/ml). For cell culture, RPMl 1640 containing 5% fetal calf serum, 10 mM HEPES buffer, 2 mM L-glutamine, penicillin (50U/ml), and streptomycin (50 pg/ml) was used. These reagents were purchased from Grand Island Biological Company (Grand Island, NY). Quantitation of J ce/l-macrophage binding. This was done as described in our previous study (1 1). PEC (1.5x 1 O6 per well) trom Listeria-immune mice were incubated (2 hr. 37°C) in 16-mm diameter tissue culture wells (Falcon No. 3008, Multiwell tissue culture plate) to allow adherence of macrophages, and then the nonadherent cells were removed by washing. The resulting confluent monolayer of macrophages was used as the binding substrate. Heat-killed Listeria monocytogenes (0.3ml of lo5to 10' bacteria/ml) was added, and the plates were centrifuged (800 X G, 5 min) and then incubated as indicated inthe various experiments. Macrophages were washed extensively to remove unboundbacteria. Control macrophages were incubated in parallel with medium alone. Listeria-immune T cells were added to the macrophage monolayers (0.5 ml containing 0.3 to 1 X lo6 cells/well), the dishes centrifuged (50 x G, 5 min, 20°C), and then incubated for 5 to 60 min at 37°C. After incubation, the plates were gently agitated, and the cells nonadherent to the macrophages were collected. These were washed, counted (adlusted to equivalent cell densities), and then their specific T cell function was measured on a new set of macrophage monolayers. The decrease in the specific functional activity of T cells recovered from the macrophage monolayers treated with Listeria relative to the activity of T cells from control macrophages was taken as a measure of specific T cell-macrophage binding. Percent specific binding was calculated as follows: Let X equal the activity of T cells nonadherent to macrophage monolayers without antigen. Let Y equal the activity or T cells nonadherent to Listeria-treated macrophagemonolayers. Then: R. 19811 HANDLING 1871 BY MACROPHAGES ANTIGEN of Listeria-macrophage interaction after simple antigen uptake is required for the generation of a T cell binding substrate. Increasing the amount of Listeria 10-fold did not overcome this requirement. This operationally defined time period of Listeriamacrophage interaction will be termed processing. To more carefully analyze the temporal requirements for processing, several experiments were performed by varying the time of Listeria-macrophage interaction. In the experiment shown in Figure 3, both the time period macrophages were exposed to Listeria before addition of T cells (processing time) MINUTES HOURS and the times of T cell-macrophage interaction (binding time) Figure 1 . a. The uptake of '251-labeledListeria monocytogenes by macrowere varied. Little or no specific binding was observed when phages is shown as a function of time. "%Listeria (0.5 ml per well, l o 5cpm per the combined binding time and processing time was less than well) was added to tissue culture wells containing macrophage monolayers formed with 1O6 PEC. The plates were centrifuged (800X G, 5 min), incubated 30 min. It appeared that the presence of T cells for various for the indicated periods of time at 37% and then washed to remove unbound lengths of time had no substantial influence on the temporal bacteria. The macrophage-associated radioactivity recovered after treatment with 1% Triton X-1 00 is shown (closed symbols). Open symbol represents '*% progression in thedevelopmentof T cell-binding substrate, Listeria associated with culture well in the absence of macrophages. b. The since the magnitude of specific binding was directly related to READOUT SYSTEM '*% natant was followed over time in culture at 37°C. The catabolism of '251-labeledListeria by macrophages was evidenced by the decrease in TCA-precipitable radioactivity associated with 0 40 0 40 80 0 40 EO the macrophage, concomitant with the release of TCA-soluble X SPECIFICBINDJNG lZ5l released into the supernatant. Less than 10% of total lZ5l Figure 2. Macrophage monolayers (MAC) were exposed (5 min, 800 x G, remained macrophage associated as TCA soluble material for 20°C) to heat-killed Listeria monocytogenes (LM) (0.3 ml of 1O6 or 1O7 bacteria ml), washed, and then incubated for 0 or 60 min at 37°C before addition of was secreted in a TCA- Tpercells several hours; less than 5% of total lZ5l (5 x 1O5 per well in 0.5 ml). T cell-macrophage interaction initiated by a precipitable form. In 10 experiments, after 1-hr culture at 37"C, brief centrifugation (50 X G, 5 min) was conducted for a total of 15 min at 37°C. Percent specific binding was monitored by the readout systems as indicated and 34.8 f 2.9% (mean f standard error of the mean [SEMI) of percent specific binding calculated as described in Materials and Methods. The the total radioactivity taken up by the macrophages was re- mean f SEM for duplicate binding reactions is shown. Control values for the leased in the mediumas small m.w. material. If the rate of activity of T cells exposed to macrophage monolayers in the absence of Listeria catabolism was measured beginning immediately after the were 12,401 f 410 Acpm for mitogen protein and 7.853 f cpm for proliferation. binding of '251-Listeriato the macrophage cell surface (a 5-min exposure instead of a 30-min exposure), catabolism was deloo Bindtng Time layed by 15 min, presumably reflecting the time required for 5min A 15 min ingestion and lysosomal interaction. Thus, the catabolism of 30 min Listeria by macrophages was an extremely rapid event, digestion being detectable within a few minutes and proceeding over several hours in culture. Catabolism did not occur at 4OC or after treatment of macrophages with paraformaldehyde (1 YO,5 min). About 50% inhibition of catabolism was observed when measured in the presence of both sodium azide (1O-' M) and 2-deoxyglucose (10" M) (data not shown). Kinetics of antigen processing. Having analyzed the kinetics of uptake, ingestion, and catabolism of Listeria by macrophages, we investigated the time of Listeria-macrophage interaction requiked to generate a substrate for T cell binding. In Figure 2, macrophage monolayers were exposed to Listeria for 5 min to allow uptake and then washed. One set was immediately tested for their ability to bind specific T cells, whereas the PUOCESShVG TIME fminl other was incubated for 60 min at 37°C before addition of T Figure 3. Macrophage monolayers were incubated at 37OC with Listeria (0.3 cells. Thus, the antigen-uptake phase (5 min) and the T cellml of lo7 bacteria per ml) for various periods of time (Processing Time)as macrophage binding reaction (1 5 min)were held constant. indicated and then washed before addition of T cells. T cells were added to the Macrophagestested for T cell binding immediately after antigen macrophages (5 x lo5T cells per well in 0.5 ml) and incubated at 37°C for the times indicated (Binding Times). The Listeria-macrophage interaction and T celluptake showed little or no specific binding, whereas those macrophage interaction were initiated by centrifugation steps as described in incubated for 60 min at37OC before the T cell-macrophage Materials and Methods. The mitogenic protein assay wasused to monitor binding: reaction showed substantial binding of T cells. Clearly, a period T cell activity for the control reaction (No Listeria) was 6002 f 353 Acpm. F Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 ingestion of Listeria monocytogenes by macrophages was monitored visually by indirect immunofluorescence with an anti-Listeria antibody. Bacteria on the macrophage cell surface but not those that are ingested are reactive with the antibody. Each point represents the number of surface bacteria per macrophage (mean f SEM). Two different experiments are indicated by the different symbols. Time zero represents macrophages exposed to Listeria for a 5-min centrifugation period (as in Fig. la), washed, and fixed immediately. c. The catabolism of Listeria macrophages was followed with time. Macrophage monolayers (1' 0 PEC per well) were exposed (30 min) to '*'I-Listeria to allow uptake and ingestion as in Figures 1a and 1 b and then incubated for various periods of time at 3 7 T . Trichloroacetic acid- (1 0%) soluble (open symbols) and -precipitable (closed symbols) radioactivity was followed in the macroqhage (triangles) and in the supernatant (circles). 1872 KIRK ZIEGLER AND EMlL R. UNANUE T I T/M€ fmiid Figure 4. The T cell-macrophage binding assay was conducted byemploying variable times of Listeria-macrophage interaction. The time indicated represents the period oftime beginning with the addition of Listeria to the macrophages and ending with the removal of T cells from the macrophage monolayers. The pooled data from 9 experiments is shown. Each point represents the mean f SEM of more than 3 separate determinations. Two experiments were carried out with a 5-min T cell-macrophage binding time, 4 with a 15-min binding time, and 3 with a 30-min binding time. Listerra monocytogenes was used at an optimal density of 10' per ml. Binding was monitored bythe mitogenic protein (3 experiments) and proliferation assays (6experiments). 127 TABLE II Effect ofmetabolic and proteinsynthesis inhibitors on antigen presentation' Binding Substrate Treatment Mitogenic Protein Activity of Macrophage Nonedherent T Cells. hcpm i SEM % Specific Blnding Expt. 1 Macrophages Macrophages and Listeria None 7.629 f 487 4,359 215 Macrophages Macrophages and Listeria 50 pg per ml cycloheximide 8.670 102 4.489 -+ 263 48 Macrophages Macrophages and Listeria 10 pg per ml cycloheximide 7,086 f 590 4,969 f 471 38 Macrophages Macrophages and Listeria 2 pg per ml puromycin 7,501 f 101 4,521 It 432 39 Macrophages Macrophages and Listeria l o - * M sodium azide 7,930 -C 373 4.025 +. 447 49 Macrophages Macrophages and Listeria 10" M 2-deoxyglucose 7,490 f 310 5.574 f 654 30 1 O-' M sodium azide 10" M 2-deoxyglucose 8.351 f 352 7,247 f 77 9 37°C 14,139 f 360 2.654 f 94 81 4°C 13.31 2 f 476 6 Macrophages Macrophages and Listeria * 42 * Expt. 2 Macrophages Macrophages and Listeria Macrophagesand Listeria Macrophage monolayers were prepared asbinding substrates by using PEC from Listeria-immune mice (1.5 x 10'PEC per well). In Experiment 1 , monolayers were incubated (30 min. 37'C) with the inhibitors at the indicated concentrations in a total volume of 0.5 ml. Heat-killed Listeria monocytogenes (50pl per well of 10' bacteria per ml) were added as indicated; the plates were centrifuged (800 x G.5 min) and then incubated for30 min at 37°C in the continued presence of the inhibitor. After Listeria-macrophage interaction,the macrophages were washed to remove the inhibitors and unbound bacteria. Listeria-immune T cells were added (5 x l o 5 per well in 0.5 ml). the plate centrifuged (50 X G.5 min). and then incubated at 37°C for 20 min. The T cells nonadherent to the macrophage monolayers were recovered, washed, counted, and then tested for activity in the mitogenic protein assay. The activity of 1 O5 cells per well is given as Acpm (mean f SEM) of duplicate binding reactions. Percent specific binding was calculated by using the activity of T cells nonadherent to macrophages that received no treatment with Listeria. The protein synthetic capacity of macrophages measured in parallel byusing 'H-leucine incorporation was inhibited over the time period of Listeria-macrophage and macrophage-T cell interaction. For example, with 10 pg per ml cycloheximide, 80% inhibition of 3H-leucine incorporation was observed. In Experiment 2, the macrophage monolayers were incubated with Listeria monocytogenes to allow uptake by macrophages and then incubated for 60 min at either 37OC or 4°C. T cells were added. and the binding reaction was carried out for 15 min at37°C. lAk reagent (neat) showed 30 to 60% inhibition. Inhibition of T cell-macrophage binding with these reagents was observed even when macrophages were exposed to the antibodies before addition of antigen. For example, whenmacrophages were exposed (30 min, 37°C) to the anti-IAk reagent, either before or after a 30-min period of Listeria-macrophage interaction, 30 f 7% or 35 f 5% inhibition (mean & SEM,3 experiments) of specific binding, respectively, was obtained. It was of interest, therefore, to address the possible role of macrophage la molecules in the handling of antigen by macrophages. In the experiment shown in Table 111, macrophages exposed to anti-la antibodies were tested for their ability to bind and catabolize '251-labeledListeria. No effect on the uptake or catabolism or antigen was observed under the conditions known to inhibit specific T cell-macrophage binding. Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 the total time of handling of the bacterium, i.e., thebinding time plus processing time. The pooled results of 9 experiments in which specific binding was measured as a function of the total time of Listeria-macrophage interaction is shown in Figure 4. The time indicated represents the period beginning with the addition of Listeria to the macrophage and ending with the removal of T cells from the macrophage monolayers. The T cell-binding ability developed after a lag period of about 20 min to maximal levels by 60 min. One-half maximal binding was observed at 45 min. In a control experiment, macrophages were incubated with Listeria for 60 min and were then given Listeria for a 2nd time immediately before addition of T cells. Specific binding was not inhibited (82% specific binding after 1 hr of Listeria vs 86% binding after the 2 exposures). We considered that the Listeria bound to the macrophage cell surface did not interfere with T cell binding. Processing requires active macrophage metabolism. If antigen handling was carried out in the presence of sodium azide (10 " M) and 2-deoxyglucose (10" M), the generation of a T cell-binding substrate was markedly inhibited (80% inhibition) (Table 11). Likewise, incubation of macrophages with bound Listeria at 4°C markedly reduced the binding of T cells (Table 11). Inhibitors of protein synthesis did not affect the binding of T cells to macrophages (Table 11). The need for metabolically active macrophages during the antigen-processing period was also apparent when testing the binding ability of paraformaldehyde-treated macrophages (Fig. 5). T cells did not bind to macrophages treated (5 min, 20°C) with 1% paraformaldehyde immediately after antigen uptake. However, substantial (approximately 60% of control) T cell binding was observed to macrophages allowed to process Listeria for 50 min at 37°C before fixation. Role of la in antigen handling. As indicated in previous studies, the exposure (30 to 60 min, 37°C) of macrophages to antibodies directed against I-region gene products resulted in inhibition of T cell-macrophage binding (1 1 ) . With an A.THanti-A.TL serum (anti-lak)at a 1:10 dilution, 70 to90%inhibition of specific binding was routinely observed; a monoclonal anti- [VOL. ANTIGENHANDLING 19811 1a73 BY MACROPHAGES READOUT SYSTEM f Treatment of Binding MITOGENIC PROTEIN PROLlF€RAT/ON CYTOTOXICITY TCElL NUMBER Macrophages ? X) 0 20 40 0 20 40 60 80 0 20 40 60 % SPECIFIC BINDING TABLE 111 lnabflity of anti-fa antibody to after the uptake or catabolfsm of antigen by macrophages" Treatment of Macrophages None Anti-IA* (undiluted), 30 min Anti-lak ( 1 :lo). 30 min % Antigen uptake" (5 Min) 30 28 33 % Antigen Catabolism' 15 Mm 50 Mln 8.3 32.721.8 7.4 33.321.6 19.8 6.5 84 Min 31.4 Macrophage monolayers were prepared from the PEC (lo6 per well) of Lfsteria monocytogenes-immune mice. Macrophages in this experiment were 76% IA-positive as judged by indirect Immunofluorescence. Macrophages were incubated (30 min, 37°C) with either a monoclonal ant"* antibody preparation or an A.TH anti-A.TL serum (anti-la!'). '251-Listefia(lo5cpm per well) was added; the plates were centrifuged (800 x G . 5 min), and then the '251-Listerianot bound to macrophages removed by washing. The macrophages bound with 1251-Listeria were then incubated for various periods of time at 37OC. and the TCA-soluble (1 0%) radioactivity released into the supernatant was assessed. Antibodies were present throughout the 3 7 T incubation period. I I ! , I Percent antigen uptake represents the percentage of added '251-Listefiathat 0 20 40 60 i o ' 100 ' 1% became macrophage associated during a 5-min exposure to '251-Listeria. TIMEfmml Percent catabolism represents the percentage of macrophage-associated radioactivity released into the supernatant as TCA-soluble material after the figure 6 . The percentage (90)antigen uptake, ingestion, catabolism, and indicated time of incubation at 37°C. recognition is shown as a function of time. Antigen uptake is shown as a percentage of'251-Listeria taken up. Antigen ingestion is expressed as the percentage decrease in the number of Listeria associated with the macrophage cell surface. Antigen catabolism represents the percentage of total '251-Listeria DISCUSSION taken up by macrophages which is secreted as TCA-soluble material. Antigen recognition represents the percent specific binding of T cells. The major thrust of this study was to analyze the kinetics of antigen uptake, ingestion, and catabolism by macrophages and to relate such events to the ability of macrophages to serve as a binding substrate for antigen-specific T lymphocytes. The system that we chose for analysis of antigen-handling events clearly involves the recognition of Listeria antigen by T cells in the context of I-region products of the macrophage. Because the specific binding of T cells to macrophages could be measured during a short (5- to 15-min) incubation period, it was possible to follow the temporal development of a T cell-binding substrate with increasing time of antigen-macrophage interactions (summarized in Fig. 6). We found that T cell binding to macrophages developed after a 30- to 60-min period of antigen handling. These kinetics were in marked contrast to the rapid uptake and ingestion of Listeria by the macrophages, indicating that antigen uptake alone-a surface event-was insufficient to generate a macrophage-associated immunogen for T cell binding. These results formed the basis for the operational definition of macrophage post-antigen-uptake events required for antigen recognition by T cells as "antigen processing." Antigen processing events may alsobe dissociated from antigen uptake on the basis of temperature and energy dependence. Although simple antigen uptake by macrophages can occur at 4OC or in the presence of inhibitors of oxidative and glycolytic metabolism, azide, and 2-deoxyglucose, little or no T cell-binding ability develops under these conditions (Table II, Fig. 6). These results are in keeping with previous studies (22, 23) which suggested that the accumulation of immunogenically relevant antigen by macrophages proceeds by membrane binding and then subsequent metabolic-dependent sequestration of bound antigen. Our findings that the ingestion of bacteria by the macrophage precedes the development of T cell-binding ability is in keeping with this pathway of antigen handling. The requirement for active macrophage events during antigen processing was also apparent when the binding ability of paraformaldehyde-fixed macrophageswas studied (Fig. 5).We conclude that once antigen processing by macrophages is Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 Figure 5. Macrophage monolayers were exposed to Listeria monocytogenes (LM) as described in Figure 4 to allow uptake and then treated as indicated in the schematic. The 1st group was treated with PES instead of 1 % paraformaldehyde (PCH20) and cultured in parallel as a positive control. All culture periods were at 37%. and treatment with PCH20 and PBS was carried out at 20°C. Extensive washing is indicated by the vertical curved line. The T cell-macrophage binding reaction was performed as described before. With each condition, binding macrophages without exposure to Listeria served as controls, no alteration in these control activities being seen with paraformaldehyde treatment. Specific binding was monitored by the indicated readout systems. The pooled results of 4 separate experiments are shown as the mean f SEM percent specific binding. 1874 KIRK ZIEGLER AND EMIL R. UNANUE 127 surface la molecules. It should be noted that studies ofthe interaction of macrophage-bound molecules in B cell-T cell interactions have provided evidence for macrophage-associated molecules, but which retained their native structure and sensitivity to trypsinization (15, 34). Thus,the macrophage apparently serves through 2 distinct pathways of antigen handling to present antigen to both major sets of lymphocytes (2, 34). The survival values of antigen degradation in the handling of pathogens may be viewed as a way to increase the number of different structural moieties that can serve as antigens. This advantage may be particularly relevant with regard to complex intracellular pathogens such as Listeria monocytogenes. Thus, bacterial components normally sequestered in the interior of organisms could conceivably serve as antigens, and the multiplicity of such antigenic determinants would make it less likely that a nonresponder status with respect to I-region gene function would be generated. Acknowledgments. The excellent technical assistance of Ms. Andrea P. Justice is gratefully acknowledged. We also thank Ms. Barbara K. Gricus for her editorial and secretarial assistance during the preparation of this manuscript. REFERENCES 1. Moller. G., ed. 1978. Role of macrophages in the immune response. Immunol. Rev. 40:l. 2. Unanue, E. R. 1981. The regulatoryrole of macrophages in antigenic stimulation. Part Two: Symbiotic relationship between lymphocytes and macrophages. Adv. Immunol. 31 : l . 3. Rosenthal, A. S . 1980. Regulation of the immune response-role of the macrophage. N. Engl. J. Med. 303:1153. 4. Shevach, E. M.. and A. S . Rosenthal. 1973. Function of macrophages in antigen recognition byguinea pig T lymphocytes. II. Role of the macrophage in the regulation of genetic control of the immune response. J . Exp. Med. 138:1213. 5. Lipsky. P. E..and A. S. Rosenthal. 1975. Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea pig lymph node lymphocytes and syngeneic macrophages. J. Exp. Med. 141: 138. 6. Ben-Sasson. S . Z., M. F. Lipscomb. T. F. Tucker, and J. W. Uhr. 1977. Specific binding of T lymphocytes to macrophages. II. Role of macrophageassociated antigen. J. Immunol. 11 0:1493. 7. Werdelin. O.,and E. M. Shevach. 1979. Role of nominal antigen and la antigen in the binding of antigen-specific T lymphocytes to macrophages. J. Immunol. 123:2779. 8. Werdelin. O., 0.Eraendstrup. and E. M. Shevach. 1979. Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers. J. Immunol. 123:1755. 9. Lipscomb. M. F., S. Z. Ben-Sasson. T. F. Tucker, and J. W. Uhr. 1979. Specific binding of T lymphocytes to macrophages. IV. Dependence on cations, temperature, and cytochalasin E-sensitive mechanisms. Eur. J. Immunol. 9.1 19. 10. Lyons, C. R., T. F. Tucker, and J. W. Uhr. 1979. Specific binding of T lymphocytes to macrophages. V. The role of la antigens on macrophages in the binding. J. Immunol. 122:1598. 11. Zlegler. K.. and E. R. Unanue. 1979. The specificbinding of Listeria rnonocytogenes-immune T lymphocytes to macrophages. I. Quantitation and role of H-2 gene products. J. Exp. Med. 150:l 143. 12. Ziegler, K.. and E. R. Unanue. 1980. Quantitation of genetically restricted T cell-macrophage binding. In Macrophage Regulation of Immunity. Edited by E. R . Unanue and A. S. Rosenthal. Academic Press, New York. Pp. 245262. 13. Geha. R. S..M. E. Jonsen, B. H. Ault, E. Yunis, and M. D. Broff. 1981. Macrophage-T cell interaction in man: binding of antigen-specific human proliferating andhelper T cells to antigen-pulsed macrophages. J. Immunol. 126:781. 14. Unanue, E. R. 1972. The regulatory roleof macrophages in antigenic stimulation. Adv. Immunol. 1 5 9 5 . 15. Unanue, E. R.. and J.-C. Cerottini. 1970. The immunogenicity of antigen bound to the plasma membrane of macrophages. J. Exp. Med. 131 :711. 16. Kolsch, E., and N. A. Mitchison. 1968.The subcellular distribution ofantigen on macrophages. J . Exp. Med. 128:1059. 17. Farr, A. G.,J. M. Kiely. and E. I?. Unanue. 1979. Macrophage-T cell interactions involving Listeria rnonocytogenes-role of the H-2 gene complex. J. Immunol. 122:2395. 18. Greenwood, F. C.,W. M. Hunter, and J. S. Glover. 1963. The preparation of ‘3’1-labeled human growth hormone of highly specific radioactivity. Eio- Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 complete, then the macrophage cell surface serves to display antigen that T cells recognize. Thus, a mechanism of T cellantigen recognition involving an active T cell-induced display of antigen previously sequestered by the macrophage seems unlikely. The abilities of aldehyde-fixed tumor cells to serve as targets for T cell-mediated cytotoxic attack (24) and aldehydefixed TNP-modified macrophages to stimulate TNP-specific T cell proliferation (25) have been noted previously. The possible role of antigen catabolism in the macrophagehandling events relevant to T cell-antigen recognition is suggested by several pieces of evidence. First, T cells did not bind to intact antigen (Table I). Second, the kinetics of antigen catabolism correlated most closely with the kinetics of development of a T cell-binding substrate (Fig. 5). Third, both the appearance of T cell-binding ability and antigen catabolism were inhibited by low temperature, metabolic energy inhibition, and aldehyde fixation. Also, in results to be published, ammonium chloride, a well-studied inhibitor of protein degradation in cultured cells, resulted in a substantial inhibition of antigen catatjolism without effect on antigen uptake or ingestion. This inhibition of antigen catabolism was associated with a corresponding inhibition of the development of T cell binding. Since inhibition of protein degradation by ammonia is thought to result from an increase in lysosomal pH, thereby inhibiting the action of acid hydrolases (26,27), and/or by inhibiting phagosome-lysosome fusion (28), these results implicate the lysosome in an intracellular pathway of antigen handling relevant to T cell-antigen recognition. Finally, we examined the possibility that I-region gene products might be involved in handling of antigen by macrophages either by serving as receptors for the initial uptake of antigen or as molecules functioning in antigen catabolism. The inability of anti-la antibodies to alter the uptake or catabolism of antigen by macrophage populations highly enriched in la-positive cells (Table 111) argues against such I-region gene involvment in initial antigen-handling events.Thus, I-region gene products may function either as receptors for fragments of antigen derived from antigen catabolism and/or as structures recognized directly by T lymphocytes. The pathway of immunologically relevant antigen handling may be envisioned as follows: Antigen interacts with the macrophage cell surface directly via trypsin-sensitive receptors (29) or by way of Fc/C3 receptors and is interiorized within phagosomes. Antigen-containing phagosomes then fuse with lysosomes, and partial degradation occurs. Some small fragments of antigen created by action of lysosomal proteinases are then released and transferred to the macrophage cell surface by a process akin to exocytosis (30). At some point after catabolism, the antigen fragments may cooperate with and/or interact with la molecules and thus form a multi-molecular antigenic structure recognized by T cells (3, 4, 31, 32). In contrast to the receptor initially involved in antigen binding, the putative membrane form of this processed antigen like la is relatively trypsin insensitive (23; Ziegler and Unanue, unpublished observations). It is not known whether the intracellular handling of antigen or the putative association of antigen fragments with la molecules is the rate-limiting step in antigen processing. In this regard, it would be of interest to determine whether antigens of minimalsize, such as the octapeptide studied by Thomas et a/. (331, would show a requirement for an antigen-processing period and ammonia effects as described here for a complex bacterial antigen. It is possible that the intracellular pathway of antigen handling may be bypassed by direct interaction of antigen fragments with macrophage cell [VOL. 1981J HANDLING ANTIGEN chem. J. 89:114. 19. Farr, A. 0 . .W. J. Wechter, J. M. Kiely. and E. R. Unanue. 1979. Induction of cytocidal macrophages following in vitro interactions between LiStefi.9immune T cells and macrophages-role of H-2. J. Immunol. 1222405. 20. 0 1 , V. T., P. Jones, J. W. Goding. L. A. Herzenberg. and L. A. Herzenberg. 1978. Properties of monoclonal antibodies to mouse lg allotypes, H-2. and la antigens. In Lymphocyte Hybridomas. Edited by F. Melchers. M. Potter. and N. Warner. Springer-Verlag. NewYork. Pp. 11 5-237. 21. Beller. D. I.,J. M.Kiely. and E. R . Unanue. 1980. Regulation of macrophage populations. I. Preferential induction of la-rich peritoneal exudates by immuno\ogicalstimuli. J. Immunol. 124:1426. 22. Ellner, J. J.. and A. S. Rosenthal. 1975. Qualitativeand immunologicaspects of the handling of 2.4-dinitrophenyl guinea pig albumin by macrophages.J. Immunol. 114:1563. 23. Ellner. J. J.. P. E. Lipsky. and A. S. Rosenthal. 1977. Antigen handling by guinea pig macrophages: further evidence for the sequestration of antigen relevant for activation of primed T lymphocytes. J. hmunol. 118:2053. 24. Bubbers. J. E.. and C. S . Henney. 1975. Studies on the synthetic capacity and antigenic expression of glutaraldehyde-fixed target cells. J. Immunol. 114:??26. 25. Thomas, D. W. 1978. Hapten-specific T lymphocyte activation by glutaraldehyde-treated macrophages: an argument against antigen processing by macrophages. J. Immunol. 121:1760. 26. Seglen, P.S.. 0. Grinde. and A. E. Solheim. 1979. Inhibitionof the lysosomal 1875 BY MACROPHAGES pathway of protein degradation in isolated rat hepatocytesbyammonia. methylamine, chloroquine. and leupeptin. Eur. J. Biochern. 9 5 2 1 5 . 27. Ohkuma. S . , and 8 . Poole. 1978. Fluorescence probe measurement of the intralysosomalpH in living cells andthe perturbation of pH byvarious agents. Proc. Natl. Acad. Sci. 753327. 28. Gordon, A. H.. P. D'Arcy Hart, and M. R. Young. 1980. Ammonla inhibits phagosome-lysosome fusion in macrophages. Nature286:79. 29. Weinberg, D. S., and E. R . Unanue. 1981. Antigen-presenting function of alveolar macrophages: uptake and presentation of listeria rnonocytogenes. J. lmmunol. 126.794. 30. Calderon, J., and E. R. Unanue. 1974. The release of antigen molecules from macrophages: characterization ofthephenomena. J. Immunol. 112: 1804. 31. Erb, P., 6. Meier, and M. Feldmann. 1979. Is genetically relatedfactor (GRF) a soluble immune response (Ir) gene product? J. lmmunol. 122:1916. 32. Lonai, P.. J. Puri. and G. Hammerling. 1981. H-2-restricted antigen binding by a hybridoma clone that produces antigen-specific helper factor. Proc. Natl. Acad. Sci. 78~549. 33. Thomas, 0.W.. K. Hsieh, J. L. Schauster. M. S. Mudd. and G. D. Wilner. 1980. Nature of T lymphocyte recognition of macrophage-associated antigens. V. Contributionof individual peptide residues of humanfibrinopeptide B to T lymphocyte responses. J. Exp. Med. I52:620. 34. Unanue. E. R. 1978. The regulation of lymphocyte functions by the macrophage. Immunol. Rev. 4O:lS. VoI 127,No. 5 . November 198l Prrnted In U S A SUBSTRATE HYDROLYSIS BY IMMUNE COMPLEX-ACTIVATED NEUTROPHILS: EFFECT OF PHYSICAL PRESENTATION OF COMPLEXES AND PROTEASE INHIBITORS' KENT J. JOHNSON AND JAMESVARANI From the Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI 48 109 ineffectivenessof fluid phase protease inhibitorsblock to Theabilityof rat leukocytestohydrolyzearadiolathe protease activityof contact-activated leukocytes may beled, surface-bound protein substrate in a solid phase can take place in the assay wasdetermined, andvarious factors that influence explain how immune complex injury presence of high concentrations of serum inhibitors. the processweremeasured.Unstimulatedleukocytes hydrolyzed verylittle substrate. Whenthe cell suspension was mixedwithzymosanparticles orincubatedwith preformed immune complexes,the amount of substrate Much interest in recentyearshasbeengenerated in the hydrolysisincreaseddramatically.Notsurprisingly,imstudy of the pathogenesis of immune complex-mediated tissue mune complexes at equivalence proved to be the most injury. Perhapsthesimplestmodel of animmune-complex effective ineliciting the response.Immunecomplexes lesion is the Arthus reaction, which is a localized necrotizing attached to the surface along with the protein substrate vasculitis, with the antigen and antibody complexing within the were able to effectively induce hydrolysis, though they the tissue injury vessel wall(1 ). Earlier studies have shown that were not as effective as immune complexes in suspenseen with the Arthus reaction is complement and neutrophil sion. Three protease inhibitors, a,-antitrypsin, an-macrodependent, with abolition of either abolishing the tissueinjury globulin, and soybean trypsin inhibitor, which were able toneutralizenearly all oftheproteaseactivityin rat (2). Speculation has therefore centered in recent years on what neutrophil lysates, were tested for their ability to inhibit immune complex-induced protein hydrolysis. It was constituents of neutrophils are responsiblefor the tissueinjury. more specifically, found that when the inhibitors were surface bound alongStudiesdone with neutrophillysatesor, are withthesubstrateprotein,theywere effective in pre- lysates of cytoplasmic lysosomal granules, reveal that they capable of damaging basement membrane preparations in vitro ventingtheneutrophilsfromhydrolyzingtheprotein. However, when the same inhibitors were present in the (3). In rabbit neutrophils the specific lysosomal constituents fluid phase, they were much less effective. The relative responsible were found to be cathepsins 0 and E, in human neutrophils, neutral proteases (4). Many other studies (5, 6) Received for publication February 10, 1981. Accepted for publication July 24, 1981. The costs of publication of this article were defrayed in part by the payment Of Dage charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by National Institutes of Health Grants HL28442. HL-26809, HL-00889, and CA-29551. haveshownthatlysosomalenzymesarereleased into the extracellular fluid during events such as phagocytosis of immune complexes.Therefore,aplausibleexplanation ofthe tissue injury seen in the Arthusreaction is the releaseof neutral proteasesfrom the neutrophils during phagocytosisofthe immune complexes in the vessel wall. It would therefore follow Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 0022-1767/a1/1275-1a75%02.00/0 THE JOURNAL OF IMMUNOLOGV Copynghi Q 1981 by The Amerlcan Associalion of Immunologists