MAURICE M. BLACK and HUDSON R. ANSLEY. From the Department of Pathology, New York Medical College, New York City

Transcription

1 ANTGEN-NDUCED CHANGES N LYMPHOD CELL HSTONES. Regonal Lymph Nodes MAURCE M. BLACK and HUDSON R. ANSLEY From the Department of Pathology, New York Medcal College, New York Cty ABSTRACT The nuclcar hstones of control and antgen-affected lymphocytcs and thymocytes were measured n terms of ther ntensty of stanng wth ammonacal-slver (A-S) and alkalne fast green (FG). Subcutaneous njecton of full strength and doubly concentrated tetanus toxod nto the upper forelmb was followed by an acute decrease n the ntensty of thc FG and A-S stanng of the thymocyte nuclear hstones. Such changes n stanng werc delayed n thc nuclcar hstones of the rcgonal axllary lymph nodes. n contrast, the njecton of a l : 100 dluton of tetanus toxod caused an acutc decrease n stanng of thc nucle of the regonal lymph node lymphocytcs but only mnmal changes n the thymocytcs. An acute decrease n stanng of the lymph node lymphocytes also occurred when heterologous serum protens were used as the antgen. Pror admnstraton of cortsonc was found to nhbt the antgcn-nduced changes n thc stanng of lymphocyte nuclear hstones. No such nhbtons occurred after the admnstraton of salne soluton. The fndngs suggcst that the nuclear hstoncs play a sgnfcant role n the mmunoogcal competcncc of lymphod cells. However, addtonal studes are needed to defne the bochemcal bass of the observed changes as well as the mechansms whereby antgen-nduced changes n nuclear hstones partcpate n the complex cellular and serologcal alteratons whch are evoked by antgens, vz. plasma cell formaton and antbody synthess. n a prevous study we found that the njecton of an antgen causes an acute decrease n the ntensty of stanng of nuclear hstones of thymocytes wth ammonacal-slver (A-S) or alkalne fast green (FG) (1). The present study s prncpally concerned wth the hstones of lymphocytes of lymph nodes dranng the ste of an antgen njecton. Observatons have also been made on the acton of cortsone on antgen-nduced changes n the hstones of lymphod cells. MATERALS AND METHODS The present study utlzed male and female CF-1 mce (Carworth Farms, New Cty, New York) weghng 15 to gm. The prncpal antgen employed was tetanus toxod (Wyeth) twce concentrated, full strength, and dluted 1:100 wth sterle physologcal salne. One-tenth of a mlllter of the approprate concentraton of tetanus toxod was njected subcutaneously nto the medal aspect of the upper forelmb. The njected mce were sacrfced at varous tmes after the njecton (30 mnutes, 1 hour, 2 hours, and 24 hours). Smears were then prepared of the thymus gland and the pslateral axllary lymph nodes. n smlar studes human serum and commercal preparatons of 16.5 per cent soluton of human gamma globuln were used as the antgen. The effect of cortsone was tested by njectng 0.1 ml (2.5 rag) of a cortsone acetate suspenson nto one 797

2 TABLE Sequental Changes n Ammonacal-Slver Stanng of Lymphod Cells after the Admnstraton of Dluted and Concentrated Tetanus Toxod njected lymph node Thymus 1:100 Concentrated 1:100 Concentrated Control mnutes q hour hours hours 0.82 =/= hours forelmb and a lke amount of 0.9 per cent sodum chlorde soluton nto the other forelmb of the same mouse. Ffteen mnutes later a 0. l-m[ dose of full strength tetanus toxod was njected nto both forelmbs. Two hours after the toxod njecton the mouse was sacrfced and smears were prepared from the axllary lymph nodes of both sdes. All the smears were ar dred at room temperature, fxed overnght n 10 per cent sodum acetateneutralzed formaln, and staned for hstones by the fast green (FG) method of Alfert and Geschwnd (2) and by the ammonaeal-slver (A-S) method developed n ths laboratory (1, 3-5), and for DNA by the Feulgen procedure. The staned nucle of small lymphocytes and mature thymocytes were then measured cytophotometrcally by means of a Canalco cytophotometer as prevously descrbed (1), except that red-senstve R-136 phototubes, kndly provded by Dr. Leonard Ornsten of Mt. Sna Hosptal, New York Cty, were used nstead of l-p-21. Measurements of the extncton values of the A-S-staned nucle were made at 480 n# (E480) and at 700 m# (E700). As reported prevously, the E480 represents the total A-S-stanable hstone, and the ET00 represents the black-stanng hstone fracton, whch s low n lysne. The samples measured from each slde ncluded to 40 nucle, selected for sphercal undstorted shape. A number of the representatve smears were also doubly staned wth A-S and the Feulgen procedure. Such preparatons were measured at 440, 560, and 700 m/~. The A-S-Feulgen (AS-F) stanng method provdes a measure of the ntensty of the A-S stanng relatve to the ntensty of the Feulgen stanng of DNA n the same nucleus. RESULTS Measurements of the Feulgen stanng of the ymphocytes and thymocytes revealed no dfference between the control and antgen-treated cells wthn the perod of observaton. The mean relatve amount of DNA per lymphocyte n the control and the antgen-treated group was and , respectvely, as determned from preparatons staned wth the Feulgen procedure above. Approxmately the same amount was found for thymocyte nucle, control and antgen-treated, prepared and staned at the same tme and n the same way. n contrast to ths constancy of DNA, the hstone content, as determned by A-S and FG stanng of the thymocytes and lymphocytes, vared n relaton to the concentraton of the antgen and the tme elapsed after njecton. The detals of such changes n hstone stanng were as follows. Axllary Lymph Nodes DLUTED TETANUS TOXOD: The acute effects of the subcutaneous njectons of varous amounts of tetanus toxod on the stanng of nuclear hstones of the small lymphod cells of the proxmal lymph node and the thymus are gven n Table and Fgs. 1 and 2. As ndcated theren, the njecton of the 1 : 100 dluton of tetanus toxod was followed by an acute decrease n the A-S stanng of the nuclear hstone of the local lymph node lymphocytes. Ths decrease was clearly apparent 1 hour after the njecton of the dluted antgen and lasted for 2 hours after the njecton. Such quanttatve changes were accompaned by a change n the A-S stanng of the lymphocytes from a yellow- sh brown to a black color. However, by 5 to 24 hours after the njecton of the dlute antgen the quanttatve and qualtatve stanng approached the prenjecton values. As ndcated, the de- crease n A-S stanng 1 hour after the njecton of dluted antgen was also accompaned by a de- crease n FG stanng of the lymphocyte hstones. 798 THE JOmNAL OF CELL BOLOGY VOLUME 26, 1965

3 Tetanus toxod 60 :00,, ', ', 40, ' 0" le ~ ~1, = / E D Q- 6O 4O Full strength % e /. k P, t f t t,,, Twce concentrated --- njected oxllary lymph nodes -- Thymus t ""e~ L" 0..c c o / ' ', // Q", ## g, ' O 12 Hstone per nucleus (A-S stanng) ~GV~E 1 ntensty of ammonacal-slver (A-S) stanng of nucle of thymocytes and regonal lymph node lymphocytes 1 hour and ~ hours after the njecton of dfferent concentratons of tetanus toxod nto the upper forelmb. The dluted antgen produces a more acute decrease n the stanablty of lymphocyte hstone than do the full strength and concentrated antgens. n contrast, the thymocyte hstones are more dstnctly depressed by the more concentrated antgen. The normal range of hstone concentraton s ndcated by the bar at lower rght of each graph. tl The antgen-nduced changes n the color of the A-S stanng are accompaned by a change n the relaton between wavelength and extncton, as shown n Fg. 3. Ths fgure also demonstrates that the decrease n absorpton at 480 m# (E480) s commonly assocated wth an elevaton n the extncton value at 700 m# (ET00). Thus, control thymocytes were characterzed by E700 values whch ranged from about 0.10 to 0.25, whereas the Ev00 values for the antgen-affected thymocytes ranged between 0.35 and t therefore appears that the nduced changes n A-S stanng are assocated wth an ncrease n the black-stan ng fracton (ETQ0) as well as wth a decrease n the stanablty of the yellow-stanng hstone fracton as determned by subtractng ET00 from E~0. t should be noted, however, that the decrease n the yellow-stanng fracton appears to precede the ncrease n the black stanng fracton. Table documents the acute decrease n the yellowstanng fracton wthout a concomtant ncrease n the black-stanng fracton. TWCE CONCENTRATED TETANUS TOXOD: n contrast to the effects of the dluted antgen, the njecton of twce concentrated tetanus toxod produced only a slght decrease n the A-S-stanable MAtmlCE M. BLACK AND HUDSON R. ANSLEV Lymphod Cell Hstones. 1. Lymph Nodes 799

4 o (-. ( o- (D,+.. ~- 0 4O - ( Thymus njected /, ' s ' L t, t [ Tetonus toxod -- full strength --- :100. t U t oxllory lymph node ' control ronge t 1.0 c 0.5 : x t~ ~)0 6~)0 7~)0 Wovelengt h (rn/~) FGURE 8 Relaton between extncton and wavelength of ammonaeal-slver-staned axllary lymph node lymphocytes shown n Fg. 4. The sold lne represents the yellowsh lymphocytes from the eortsone- "protected" sde; the dashed lne s the curve of the black-stanng antgen-affected lymphocytes. 0 ~' o ',," Hstone per nucleus (FG stonng) FGURE ~ Relatve amounts of fast green (FG)- stanable nuclear hstones of thymocytes and regonal lymph node lymphocytes 1 hour after the njecton of full strength and dluted tetanus toxod. Note that the dluted antgen produces a greater decrease n the lymphocyte hstone than does the full strength antgen, confrmng the changes depcted n Fg. 1. nuclear hstone wthn the 2-hour perod after the njecton. At the 5-hour perod, however, the A-S stanng was decreased and changed n color from a yellowsh brown to blacksh. Twenty-four hours after the njecton, the control type of stanng was almost completely reganed. Thus the twce concentrated antgen produced a delay n the response as compared wth the dluted antgen. After ths delay, however, the antgen-nduced changes were smlar. FULL STRENGTH TETANUS TOXOD: Asndcated n Fgs. 1 and 2, only mnmal decreases n A-S and FG stanablty of local lymphocyte nucle followed the njecton of full strength antgen wthn the frst hour. Wthn 2 hours after the njecton, however, the stanablty fell to values whch were smlar to those obtaned after the njecton of the dluted antgen. That s, the results were ntermedate between those followng the concentrated and the dluted tetanus toxod. TABLE Relatve ntensty of Ammonacal-Slver-Feulgen Stanng of Control and Tetanus Toxod-Affected Thyrnocytes (l) (2) Group E440/F ET00/F () -- (2) Control Tetanus toxod 1 : mnutes hour Tetanus toxod full strength 30 mnutes O hour Thymus DLUTED TETANUS TOXOD: n contrast to ts effect on the axllary lymph nodes, the njecton of the dlute antgen produced a mnmal decrease n the ntensty of A-S and FG stanng of the thymocyte nucle and no change n the qualty (yellowsh brown) of A-S stanng throughout the perod of study. The dfferences between the responses of the thymocytes and the lymph node lymphocytes are recorded n Fgs. 1 and 2. The stanng of the thymocyte nucle was greater than that of the lymphocyte nucle, 1 and 2 hours after the njecton of the dluted antgen. CONCENTRATED TETANUS TOXOD: Wthn 30 mnutes after the njecton of the concentrated 800 Tn~ JOURNAL OF CELL BOLOGY VOLUME ~6, 1965

5 antgen, the A-S stanablty of the thymocyte nuclear hstone decreased almost 30 per cent and the color of the stanng became blacksh. By 1 and 2 hours after the njecton, the A-S stanablty had contnued to decrease whle the FG stanablty fell approxmately per cent below the control values. Such changes n the thymocytes followng the njecton of the concentrated antgen were n contrast to the mnmal changes n the nucle of the lymph node lymphoeytes. The stanablty returned toward the control values, however, by 5 to 24 hours. FULL STRENGTH TETANUS TOXOD : The effects of the full strength dosage werc smlar to, although less markcd than, those produced by the concentrated antgcn. Wth both these dosagcs the ntensty of stanng of thymocytc hstoncs 1 hour after the njecton was lower than that of the lymph node lymphocytes, and remaned dcpressed wth lttle change at the 2-hour perod. t wll be noted that at the 2-hour mark the stanng of the lymphocytcs of the mce njected wth the full strength antgen was smlar to that of the thymocytes from the same mcc. On the other hand, thc ntensty of stanng of the thymocytes from mce njected wth the doubly conccntratcd antgen was lower than that of the correspondng lymphocytes. HETEROLOGOUS SERUM : One hour after the njecton of 0.1 ml of human serum nto the forelmb of a mouse there was a decrease n the A-S stanng of nucle of the lymphod cells of both the thymus and the dranng axllary lymph nodes. The qualtatve and quanttatve changes n the lymphocytes and thymocytes were smlar to those occurrng 2 hours after the njecton of full strength tetanus toxod. Smlar effects were produced by the njecton of 0.1 ml of a 16.5 per cent soluton of human gamma globuln. Whereas a 2-fold ncrease n the tetanus toxod caused a delay n the effect on the nuclear hstones, no such delay was found after the admnstraton of a smlarly ncreased dosage of heterologous proten. PROR NJECTON OF CORTSONE : The njecton of 9.5 mg of cortsone nto the upper forelmb, 15 mnutes before the njecton of full strength tetanus toxod, was found to prcvcnt the changes whch the antgcns causcd n thc hstoncs of the lymphocytes of the axllary nodes. After the A-S and FG stanng, the lymphocytes from such nodes appeared smlar to control lymphocytcs. n contrast, howevcr, the lymph nodes njectcd wth salne nstead of cortsone, before the njecton of tetanus toxod, exhbted all the antgen-nduced changes n ther nuclear hstones. Such lymphocyte nucle revealed a decrease n the ntensty of stanng by A-S and FG, and showed a blacksh color wth A-S. The dfferences n the effect of the pror njecton of cortsone and salne on the antgen-nduced changes n lymphocyte nuclear hstones are documented n Fg. 4. Ths fgure demonstrates that the ntensty of stanng of the hstone of the salnetoxod-treated lymphocyte nucle was smlar to that of lymphocyte nucle after toxod alone. n contrast, the A-S and FG stanng of the cortsonetoxod-treated lymphocytes was smlar to that of control lymphocytes. A-S--FEULGEN STANNG : Measurements of the AS-F-staned cells confrmed the changes demonstrated by measurements of the nucle staned wth A-S alone. As ndcated n Table, antgen njecton was followed by a decrease n the ntensty of E440/F rato. Ths decrease was due to a decrease n the yellow-stanable hstorc (E44o mnus E7oo). n contrast, the EToo/F rato tended to ncrease. COMMENTS The present study has confrmed our prevous observatons that the njecton of an antgen has the effcct of nducng an acute reversble decrease n the A-S and FG stalnablty of nuclear hstones of lymphod cells of the thymus and lymph nodes. t has also been observed that the effect on the local lymph node lymphocytcs s less acute after the admnstraton of full strcngth and doubly conccntratcd tetanus toxod than after 1 : 100 dlutons of tetanus toxod. Ths delay s suggestve of the phenomenon of mmunologcal paralyss, wheren a hgh dose of an antgen may produce tolerance rather than antbody producton. n ths connecton t should be rccalled that hstoncs tend to form prccptatcs wth acd protens. However, such precptates usually dssolve n the prcsencc of excess hstone. t would bc of ntercst to know whether a smlar stuaton obtans n thc reacton between antgens and the hstones of lymphod cells. That s, does antgen excess lead to the formaton of nsoluble antgcn-hstonc complcxes wth resultant mmunologcal ncompctcnce? The lack of such a "paralyss" cffcct on the thymocytc hstoncs mght reflect ntrnsc dffercnccs between thymocytcs and lymphocytcs n rcgard to ther response to antgcns. Such dffcrcnccs are sug- MAmlCE M. BLACK AND HUDSON R. ANSLEY Lymphod Cell Hstones.. Lymph Nodes 801

6 60 50 FG t~ Tetanus toxod -- plus cortsone --- plus salne 40 a # 30 t 0 C (3" 4, , _ 30 A-S A 0 / /.'" / ~ ", Hstone per nucleus FmunE 4 Fast green (FG) and amnmnaeal-slver (A-S) stanng of hstones of lymphocytes from rght and left axllary lymph nodes of the same mouse ~ hours after the njecton of cortsone and tetanus toxod nto the left forelmb and salne and tetanus toxod nto the rght forelmb. Note that the sde exposed to salne and toxod shows the usual antgen-nduced decrease n FG- and A-S-stanable hstone. No such change occurs on the cortsone-"protected" sde. gested by the fact that the color of A-S-staned axllary node lymphocyte nucle tends to be even more yellowsh than that of thymocyte nucle, thus ndcatng a somewhat dfferent hstone tampa- ston. Nevertheless, the probablty that the dfference s a functon only of the concentraton of the antgen reachng the cels s supported by other studes wheren we observed that the njec- 802 THE JOURNAL OF CELL BOLOGY VOLUME ~6, 1965

7 ton of concentrated tetanus toxod nto a hnd foot pad of a gunea pg caused an acute decrease n the A-S stanng of the ngunal lymph node lymphocytes, whle the lymphocytes of the poplteal nodes were unchanged. t would seem that further nvestgatons are needed n order to defne the detaled relaton between antgen concentraton and the nuclear hstones of dfferent types of lymphod cells. t s well known that cortsone may depress mmunologcal competence and exert a lymphocytolytc effect. t was therefore of nterest to fnd that cortsone nhbted the antgen-nduced decrease n hstones n lymphod cells. Such fndngs would suggest, though t remans to be proved, that the antgen-nduced changes n the nuclear hstones of lymphod cells are related to mmunologcal competence. The data do not permt a defntve explanaton of the bochemcal bass of the antgen-nduced changes n the FG and A-S stanng. The decrease n stalnablty mght represent an actual loss of hstones from the nucle of lymphod cells. Such a lberaton of hghly reactve molecules mght bnd antgens n a non-specfc fashon or serve as nstructonal materal f taken up by retcular cells. On the other hand, the data mght also be explaned by a change n the reactve groups of the hstones wthout any dscharge of nuclear hstone. t wll be recalled that the FG stanng and partcularly the A-S stanng depend on the presence of free e-amno groups of lysne. Blockng of such groups by acetylaton, deamnaton, or combnaton wth non-hstone protens (antgen?) could decrease the stanablty of such altered hstones. t should be mentoned that the pror treatment of lymphod cells wth ntrous acd (HNO2) causes a loss of subsequent yellow A-S stanng wth an accompanyng decrease n the E~0 value but wthout any apprecable effect on the ET00 value. Smlar observatons have been made n regard to the A-S stanng of solated thymus hstones on cellulose acetate strps; that s, the "loss" of the yellow-stanng hstone s not necessarly assocated wth a concomtant transformaton to black-stanng hstone. Measurements of dry mass of the nucle by means of nterferometry mght prove rewardng n determnng whether the observed changes nvolve an actual loss of proten from the nucleus. t should be noted, however, that ether the loss of hstones or the neutralzaton of hstone amno groups may alter the RNA synthetc functon of DNA (7). t s nterestng to speculate on the queston whether the observed changes n hstones play a role n the transformaton of lymphod cells nto antbody-producng cells or whether RNA produced by lymphocytes may be transferred to retcular cells whch n turn acqure the ablty to produce antbody and are transformed nto plasma cells. Though the avalable data are ncomplete, they suggest that the antgen-nduced changes n the lymphod cells play a sgnfcant and a htherto unsuspected role n the cellular and humoral response to antgen. Ths nvestgaton was supported by Unted States Publc Health Servce grant CA from the Natonal Cancer nsttute. Receved for publcaton, February 26, REFERENCES 1. BLACK, M. M., and ANSLEY, H. R., Antgennduced changes n lymphod cell hstones.. Thymus, J. Cell Bol., 1965, 26, ALFERT, M., and GESCHWND,., A selectve stanng method for the basc proten of cell nucle, Proc. Nat. Acad. Sc., 1953, 39, BLACK, M. M., SPEER, F. D., and LLLCK, L. C., Acd extractable nuclear protens of cancer.. Stanng wth ammonacal-slver, J. Nat. Cancer nst., 1960, 25, BLACK, M. M., and ANSLEY, H. R., Hstone stanng wth ammonacal-slver, Scence, 1964, 143, BLACK, M. M., Hstorc stanng, Methods mmunol., n press. 6. BLACK, M. M., and ANSLEY, H. R., nduced alteratons n nuclear hstones, Fed. Proc., 1965, 7. ALLFREY, V. G., Nuclear rbosomes, messenger- RNA and proten synthess, Exp. Cell Research, Suppl., 1963, 9, 183. MAURCE M. BLACK AND HUDSON R. ANSLEY Lymphod Cell Hstones.. Lymph Nodes 803