Scand. J. Immtotot. 8, 515-524, 1978
Destruction of Sensitized Erythrocytes by Human Monocytes in Vitro: Effects of Cytochalasin B, Hydrocortisone and Colchicine A. FLEER.' M. L. J. VAN SCHAIK.^ A, E, G. KR. V O N DEM BORNE'-^ & C. P. ENGELFRIET* ' Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Immunohaematology. Amsterdam. ' Pediatric Clinic. Binnen Gasthuis, Amsterdam, and ^ University Clinic of Internal Medicine. Department of Hematology, Binnen Gasthuis. Amsterdam, The Netherlands
Fleer, A,, van Schaik. M.L.J.. von dem Borne, A.E.G.Kr. & Engclfrici, C.P. Destruction of Sensitized Erjthrocytes by Human Monocytes in Vitro: EITects of Cyiochalasin B. Hydrocorlisonc and Colchicine, Scand. J. Immunol. 8. 515-524. 1978. The purpose ofthis siudy was lo characterize ihe destruction of sensitized erythrocytcs by human blood monocytes in viiro. The incubalion in vilro of human monoeyies with "Cr-labelled human erylhrocytcs sensitized with IgG rbesus alloantibodics anti-D (EAIgO anli-D) resulted in release ol'^'Cr from the crythrocytes (lysis) as well as uptake of^'Cr-labclled erythrocytes by the monocytes (phagocytosis). The lysis or E A IgG anti-D by monoeyies was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocorlisonc and colchicine inhibited lysis, bul had no effect on phagocytosis. These agents did not afTect binding of EAIgG anti-D to monocytes. The efl'ect of these agents on lysis corresponded 10 their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was. likewise, enhanced by cytochalasin B and inbibiled by hydrocortisone and colchicine. A significant correlation was found between lysosoma! enzyme release and lysis. Together, tbese resulls strongly suggest tliat lysosomal enzymes, released by the monocytes when ineubaled with anti-D-sensilized erylbrocyies, are responsible for the cytotoxic activiiy of these cells towards sensitized erythrocyies. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erjtbrocyte, because only erythrocyies tbat were bound to ihe monocytes were lysed. Tbe finding of other investigators tbat removal of monocytes from suspensions of human mononuciear leucocytes results in a strong reduction in tbe cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro, was confirmed. C. F. Engelfriet, Central Laboratory of the Netherlands Red Cro.i.i Blood Transfusion Service. F.O. Box 9190. Amsterdam. The Netherlands.
In patients with autoimmune haemolytic anaemia (AIHA) due to non-complement binding IgG autoantibodies. erythrocytes are predominantly cleared by and destroyed in the spleen [6]. It is generally assumed that mononuclear phagocytes, located in the spleen, are primarily responsible for the elimination and destruction of erythrocytes in patients with this type of AIHA [8], 0300-9475/78/1200-0515 $02.00
In vitro, monocytes are capable of lysing hutnan erythrocytes sensitized with IgG anti-D [17], IgG anti-A [18] and IgG anti-B [31] alloantibodies, as well as with heteroantibodies [42]. The mechanism of lysis is still unclear, although Holm [16] has provided evidence thai lysis of erythrocytes sensitized with IgG anti-A (EAIgG anti-A) by monocytes is due to phagocytosis and intracellular digestion of such cells.
1978 Blackwell Scientific Publications
515
516
A. Fleer etal.
In contrast, animal macrophages are capable of lysing EAIgG in hcierologous systems by nonphagocytic mechanisms, i.e. extracellular lysis of EAIgG 114. 35. 37.40]. The contribution of these mechanisms to destruetion of EAIgG anti-D by human monocytes in vilro is not known, bui phagocytosis is not always observed [ I . 2. 19, 23. 32]. Insight into this niechanism is essential, since this system constitutes a model in vitro of the destruction of erythrocytes in vivo in patients with AIHA due to non-complement binding IgG autoantibodies. Serologically and immunochemically such antibodies closely resemble rhesus antibodies and regularly show rhesus specificity [8]. In the present paper the resulls of studies on the mechanism of destruclion of anti-Dscnsitized erythrocytes by human monocyles in vitro are described.
MATERIAL AND METHODS Donors. Venoui blood was collected rrom heallhy donors. .Sera. Fclol culf .scrum (FCS) was obtained from CJihco Biocult. Olusgow. Scoitund, Anii-D sera were ohtuincd from donors who had been immunized against the Rh(D) untigen hy repeated intravenous injections with OR,r erythrocytes. The tiire of ihesc anir-D alloantisera in the indirect aniiglohulin lest varied from 1:2000 to I:I6.(KX> when lestetl with rubbit amihuman globulin serum. Agents. Cyloctiul;isiii B was a product of ICI, Aldcrlcy Park. Cheshire. England, It was dissolved in dimethylsulphoxide (DMSOl to u cuiicenlration ol' 3 mg ml. This solution was further diluted in phosphaicbutTered saline (PBS) lo a conccniraiion of cytochalasin B of SO ^g ml (concentration of DMSO. I" „} and stored at T-4 C, Colchicine was purchased from Mcrcl. AG. Darmstadt, West Germany. Hydrocortisone sodium succinnte wa.s u gifl from Ortfunon, Oss. The Netherlands, Preparation n/ monoiyits. Mononuciear cells were prepared by spinning peripheral blood o%er FicollIsopaque (density 1.077 g,'cm* at 25 C), as described by Loos el at. |24], The resulting intcrfucc layer, whicb contained the mononuciear cells, was washed three limes in minimtil essential medium (MFM) (Gibcu. Grand Islimd. N,Y., USA) supplemented wiih 10",, (V V) FCS. Hcnccfurlh. this medium will be called MEM 10°, FCS. After being washed, the mononuclcar cells were resufpended in MEM 20". FCS to a fmal concentration of 10* celb;ml. This suspension wa* layered on plastic Petri dishes lOptiUix. Falcon Plastics. Oxnard. Calif,, USAt. 2.5 ml per dish, Afler incubalion Ibr 11 h al 37 C, ilic plutcs were vigorously washed to remove the non-adherent cells. The adherent cells were
carefully scraped olt wnli a piece ofsiliconc rubber and resuspended In M I M 10",. FCS. Finally, the cells were washed once, and the cell coneenlralion was adjusted as indicated in Results, The average percentage of monocytes in the adherent cell preparations was 71 I.Tp (mean • SD of twenty donors), as judged by morphology and si/e distribution |25|, Frvf.araiion. labelling and.n-nsitiration o/ eryihrocyte A 5",, suspension of O. rhesus positive (type R^Rj) erythrocyies from freshly drawn heparini/.cd venous blood was prepared in a storage medium containing 2',. (w/vl BSA (bovine serum albumin). The ervihrocyte suspensions were used for 1-2 weeks. Before use the cr>tbrocytes were washed three times in PBS and resuspended in MEM 10"„ FCS to a conccntraiion of 2 IO»;ml. Labelling of erythrocytes. For labelling, equal volumes of erythrocyie suspensions (W/ml) and an Na^^'CrOi solution (Radiochemical Centre. Amersham. England; specitic aaivity 50-400 mCi/mg Cr) were mixed and incubated for 1 2 h at 37 C. The ery ihrocytes were ihen washed three times in MEM 10" „ FCS and were subseiiuenily resuspended to a concentration of I -32 • 10* cells,'ml. Sensitizatiiin itf eryihrocytes. Erythrocytes were sensitized with anti-D tiy mixing ihe erythrocyte suspension 12- 10* mil with an equal volume of undiluted anii-D serum. Subsequently, the eryihrocyte suspension was incubated witb " C r and trealed as described above under 'Labelling of erjihroeyies". Cytutaxicity assay. The cytotoxicity assay was performed as described by Zeijlemaker et al. [421. Briefly. SOij.1 of monocyic suspension (IO"/mll, 50 |/l of anti-DsensitiMd eryihrocytes (l-33> lO"/ml) and 50 \L\ of MFM I0"o FCS were added to wells of round-bottomed microtitre plates. Afler incubation for 16 b at .17 C the plates were centrifuged al 500 g for 10 min at room temperature. A IOO fil sample was taken from the supernatant of each well to determine the percentage of *'Cr release, Specitic cytotoxicily or specific lysis was defined as: E-S
lotal cpm m which £is experimental *'Cr release (monocytes wiih EAIgG anti-D(, S is spontaneous " C r release (monovytes with non-sensiti/ed erythrocytes) and total cpm (counts per ininuti') is total radioactivity per 50 jil erythrocyie suspension. Fach experimeni was performed in triplicate. From tlie percentage of specitic cytotoxicity the absolute number of erythrocytes lysed was calculated by multiplying this percentage by Ihe number of eryihrocyies per well Colchicine and hydrocortisone were dissolved in MFM \Q'\ FCS and added directly lo the wells. Cytochalasin B (50iig/ml in PBS • ! " „ DMSO) was diluted III MEM I O \ r e s and added to the wells to a tinal concentration of ( ixg,ml. The corresponding concentration of DMSO (0.02".. v v» had no influence on cyioioxicity. Fhagoeytosis assay. Phagocytosis was determined from tbe same wells in which lysis ("Cr release) was measured. All bul 25 [xl of the supernatant was carefully removed. Next, 100 [xl distilled water were added
Mechanism of Monocyte Cytotoxic Activity to the wells, and the cell mixture was stirred and incubated for 10 min ai room lemperaiure. Thereafier, the plates were centrifuged. and iOO [j.1 of the supernatant were removed and counted Cor radioactivity. The cell pellets were dissolved in 10% saponine and counted separately Tor radioactivity. The counts of ihe pellet Traction were corrected for ihe 25 [i.1 of ihe "waler" supernatant that remained in the pellet to yield ihe nel cpm of ihe pellet. Phagocytosis was defined as: net cpm pellet — X 100% total cpm Specific phagocytosis was defined as percentage phagocytosis of EAlgG anti-D minus percentage phagocytosis of non-sensitized erythrocytes. From ihe percentage of specific phagocytosis ihe absolute number of erythrocytes ingested was calculated by multiplying ihis percentage by the number of erythrocytes per well. Adherence assay. To determine the number of monocyies binding sensitized erythrocytes. IOO [il of monocyte suspension (2.5 v 10*/ml) were mixed with 50 [xl of a suspension of anti-D-sensitized erythrocyles (2.5-lOx lO'/ml) and 150 |j.l of medium (MEM 10% FCS). The whole was mixed and centrifuged at 150 ^ for 10 min al room temperature. The pellet was carefully resuspended and incubated for I min at room temperature wiih 25IJL1 of a solution containing acrldine orange (4 jAg/mi). The perceniage of roselte-forming monocytes (those that had bound three or more eryihrocytes) was determined with fluoresceni microscopy. Roselte formation with non-sensilized erythrocytes was 0-3%. Release of lysosomal enzymes by manocyles. Monocytes (2x 10') were preincubated for 10 min at 37'C (with or without additions, as indicated). Nexi. either anti-D-sensitized or non-sensitized erythrocyles (4 x 10") were added, and the mixture (volume^ 1.0 ml) was incubated for 60 min at 37'C. Samples of 450 fil were laken al /=Oand /=60 min and centrifuged; the supernatant was collected and Immediately placed on ice. In these supernatants the activities of ihe lysosomal enzymes lysozyme and p-gluciironidase and of the cytoplasmic marker enzyme lactate dehydrogenase (LDH) were determined as described by Goldstein er al. [10]. LDH release was used as an indicator of cell viability.
To determine Ihe lotal activity of these enzymes in the monocytes. the cells were exposed lo 0.2"n (v/v) Triion-X-IOO. The percentage specilic release of these enzymes was calculated by subtracting the percentage release from monocytes incubated wiih non-sensilized erythrocytes from the percentage release from monocytes incubated wiih sensitized erythrocytes. Srarislics. Correlations between measurements were assessed by the rank correlation meihod of Spearman [36].
RESULTS The relation between cytotoxic activity and percentage of monocytes in human mononuclear leucocyte .suspensions The monocyte content and cytoloxic activity of plastic-adherent, non-adherent and ironcarbonyl-treated (for method see Ref. 42) mononuclear leucocyte suspensions arc shown in Table I. It is evident that depletion of monocytes results in an almost complete loss of cytotoxic activity of the mononuclear cells towards EAIgG anti-D. Contribution of lysis and phagocytosis to the destruction of EAlgG anti-D by monocytes The relative contribution of lysis (specific ''Cr release) and phagocytosis (specific *'Cr uptake by monocytes) to the total number of EAlgG anti-D destroyed by monocytes was markedly influenced by the EAlgG to monocyte ratio (Fig. 1). When 5x10^ monocytes were incubated with increasing numbers of EAlgG, the lysis levelled off at an EAlgG to monocyte ratio of 2:1 to 4:1. In contrast, phagocytosis of EAlgG anti-D. negligible at
TABLE T, Relation between cytotoxic activity and percentage of monocytes Jn mononuclear leucocyte suspensions Lysis of EAlgG anti-D (number lysed •; 10=") Mononuclear leucocytes
Adherent Non-adherent Iron-carbonyl-treated MN
517
Percentage monoeytes
1:1»
20:1*
71.2±4.9t 5.9 ±0.9 5.1 ±1.7
26.3 ±2.2t 3.2 + 1.3 2.8 ±0.7
36.2 ±5.5 5.4 ±1.7 5.6 ±1.3
• EAlgG to mononuclear leucocyte ratio. To 5x 10* mononuciear cells 5x 10* or 10" HAIgG were added. The incubation time was 16 h. t Mean + SEM (/i=6).
518
A. Fleer era!. EAH)C
tm • EAlgG proc«ss«d
BO
«0
20
20
ihsgocylosis
05
1
J 4 8 no. EAlgG pof monocylo
16
33
FIG. I. Lysis and phagocytosis of EAlgG anii-D by human monocytes in vitro. Monocytes (5 > 10*) were incubiited wiih variou.s numbers of HAlgG anti-D (5x 10' to 1.6 • IC), resulting in EAlgG to monocyte raiios of 0.5:1 to 32:1 (abscissa). Ordinaie: number of EAlgG processed (i.e. lysed or phagocyti/edl ( • 10^). Incubation time: 16 h. The data shown are averages {mean ±SEM) of experiments with monocytes from eighl donors. • , Lysis of EAlgG; • . phagocytosis of EAlgG; A. toiiil number of EAlgG processed (lysis i phagocytosis).
FIG. 2. Eftect of cytochalasin B on lysis and phagocytosis of EAlgG anti-D by human monocyies. To 5 - 10' monocyies various numbers of EAlgG were added, resulting in EAlgG to monocyle ratios of 0.5:1 10 32:1 (abscissa). Cytochalasin B was added at zero time. Ordinaie: number of EAlgG processed ( • lO^I. Incubation lime: 16 h. The data presented are averages from experiments with moncyles from three donors. • , Lysis of EAlgG; o. lysis of EAlgG in the presence of cytochulasin B (1 pig/ml); • . phagocytosis of EAlgG; I i, phagocytosis of EAlgG in the presence ofcytochaiasin B (1 jig/ml); A, total number of EAlgG processed (lysis ' phagocytosis) in the absence of cy'ochalasin B.
EAlgG lo monocyte ratios of 8:1 or lower, increased at higher ratios. Effects ofcytochaiasin B on lysis and phagocytosis To determine whether monocyies were capable of lysing EAlgG independently of phagocytosis, experiments were performed with cytochalasin B, an inhibitor of phagocytosis [7, 10, 28, 27]. Fig. 2 shows that cytochalasin B (1 M-g/ml) completely blocked phagocytosis, but enhanced the lysis of EAlgG. Moreover, the ntimber of erythrocytes lysed in the presence of cytochalasin B was clearly higher ihan the total number processed in the absence of this agent (the sum of lysis and phagocytosis). This indicates that the enhancement of lysis by this
agent was not only a result of inliibition of phagocytosis. Cytochalasin B is known to enhance the release of lysosomal enzymes by phagocytic celts [7, 10. I I . 13] and its effect on the extracelltilar lysis of EAlgG suggests that lysosomal enzymes released by the monocytes may be responsible for lysis.
Effects of hydrocoriisone and cotchicine on lysis and phagocytosis To find further support for the hypothesis that lysosomal enzymes released by the monocytes are responsible for lysis of erythrocytes.
Mechanism of Monocyte Cytotoxic Activity experiments were carried out with hydrocortisone and colchicine. These agents have been shown to inhibit lysosomal enzyme release by phagocytic cells [ 12, 15, 33, 41. 43]. Fig. 3 shows that both agents inhibited lysis but had no such effect on phagocytosis.
519
Is binding of EAlgG to monocytes required for the occurrence of lysis?
To investigate the requirement of EATgG binding to monocytes for lysis the following experiment was performed: monocytes were incubated with a mixture of non-labelled anti-Dsensitized and "'Cr-labelled non-sensitized erythrocytes. Table III shows that monocytes Relation of the release of lysosomal enzymes by selectively lysed the sensitized erythrocytes, monocytes to lysis whereas the non-sensitized erythrocytes were left unaffected. In a second type of experiment, To obtain quantitative data on the relation monocytes were incubated with increasing between the release of lysosomal enzymes and numbers of non-labelled anti-D-sensitized erylysis of EAlgG by monocytes, these processes throcytes. To these mixtures a small number of were studied in parallel with monocytes of eleven '••Cr-Iabelled EAlgG anti-D C'Cr-EAlgG) were donors. The results are shown in Table II. added. Table III shows that lysis of "^'Cr-EAIgG A highly significant correlation was found decreased when the number of non-labelled between the release of lysozyme and lysis EAlgG per monocyte increased. When mono(r = 0.834; 0.001 > P>0.0005). The correlation cytes and non-labelled EAlgG were centrifuged between release of P-glucuronidase and lysis (5 min at 100 g) before the addition of "Crwas less marked, but still significant (r = 0.620; EAlgG (to establish binding of non-labelled 0,025 > P > 0.01). Table 11 also shows that the EAlgG to the monocytes), this decrease was efFects of cytochalasin B, hydrocortisone and even more marked. Supernatants of mixtures colchicine on the release of lysosomal enzymes of monocytes and EAlgG anti-D (harvested corresponded to their effects on lysis.
PHAGOCYTOSiS
CYTOTOXIC LYSIS EAlgG processed (x10^) 60
€05
40
20 HC1
16 32 1 2 no. EAlgG per monocyte
4
16
32
FIG. 3. EfTccts of hydrocortisone and colchicine on lysis and phagocytosis of EAlgG anti-D by human monocytes. 5x 10' monocyies were incubated with various numbers of EAlgG anti-D, resulting in EAlgG to monocyte ratios of 1:1 to 32:1 (abscissa). Colchicine and hydrocoriisone were added at zero time (final concentrations of I and 5 mM). Ordinate: number of EAlgG processed (x 10*). Incubation lime: 16 h. Data are averages from experimenis with monocytes from three donors. Lcfl: Lysis: A, Control; • , hydrocortisone (! mM); • , hydrocoriisone (5 mM); O, colchicine (t nm); LJ, colchicine (5 niM). Right: Phagocytosis: Symbols the same as with lysis.
520
A. Fleer et at. TABLE II. Lysosomat enzyme release by monocytes: relation lo lysis of EAlgG by monocytes*
Lysosomal enzyme release ("„) Addition Lysozyme Nil(n-ll) I fig/ml cylochalasin B in -6) 5 niM hydrocoriisone (« = 5) 5 mM colchicine (« —5)
fl-Glucuronidase
10.4 r 1.8t
3.8-0.6
18.1,; 4.1 (/'
Destruction of Sensitized Erythrocytes by Human Monocytes in Vitro: Effects of Cytochalasin B, Hydrocortisone and Colchicine A. FLEER.' M. L. J. VAN SCHAIK.^ A, E, G. KR. V O N DEM BORNE'-^ & C. P. ENGELFRIET* ' Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Immunohaematology. Amsterdam. ' Pediatric Clinic. Binnen Gasthuis, Amsterdam, and ^ University Clinic of Internal Medicine. Department of Hematology, Binnen Gasthuis. Amsterdam, The Netherlands
Fleer, A,, van Schaik. M.L.J.. von dem Borne, A.E.G.Kr. & Engclfrici, C.P. Destruction of Sensitized Erjthrocytes by Human Monocytes in Vitro: EITects of Cyiochalasin B. Hydrocorlisonc and Colchicine, Scand. J. Immunol. 8. 515-524. 1978. The purpose ofthis siudy was lo characterize ihe destruction of sensitized erythrocytcs by human blood monocytes in viiro. The incubalion in vilro of human monoeyies with "Cr-labelled human erylhrocytcs sensitized with IgG rbesus alloantibodics anti-D (EAIgO anli-D) resulted in release ol'^'Cr from the crythrocytes (lysis) as well as uptake of^'Cr-labclled erythrocytes by the monocytes (phagocytosis). The lysis or E A IgG anti-D by monoeyies was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocorlisonc and colchicine inhibited lysis, bul had no effect on phagocytosis. These agents did not afTect binding of EAIgG anti-D to monocytes. The efl'ect of these agents on lysis corresponded 10 their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was. likewise, enhanced by cytochalasin B and inbibiled by hydrocortisone and colchicine. A significant correlation was found between lysosoma! enzyme release and lysis. Together, tbese resulls strongly suggest tliat lysosomal enzymes, released by the monocytes when ineubaled with anti-D-sensilized erylbrocyies, are responsible for the cytotoxic activiiy of these cells towards sensitized erythrocyies. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erjtbrocyte, because only erythrocyies tbat were bound to ihe monocytes were lysed. Tbe finding of other investigators tbat removal of monocytes from suspensions of human mononuciear leucocytes results in a strong reduction in tbe cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro, was confirmed. C. F. Engelfriet, Central Laboratory of the Netherlands Red Cro.i.i Blood Transfusion Service. F.O. Box 9190. Amsterdam. The Netherlands.
In patients with autoimmune haemolytic anaemia (AIHA) due to non-complement binding IgG autoantibodies. erythrocytes are predominantly cleared by and destroyed in the spleen [6]. It is generally assumed that mononuclear phagocytes, located in the spleen, are primarily responsible for the elimination and destruction of erythrocytes in patients with this type of AIHA [8], 0300-9475/78/1200-0515 $02.00
In vitro, monocytes are capable of lysing hutnan erythrocytes sensitized with IgG anti-D [17], IgG anti-A [18] and IgG anti-B [31] alloantibodies, as well as with heteroantibodies [42]. The mechanism of lysis is still unclear, although Holm [16] has provided evidence thai lysis of erythrocytes sensitized with IgG anti-A (EAIgG anti-A) by monocytes is due to phagocytosis and intracellular digestion of such cells.
1978 Blackwell Scientific Publications
515
516
A. Fleer etal.
In contrast, animal macrophages are capable of lysing EAIgG in hcierologous systems by nonphagocytic mechanisms, i.e. extracellular lysis of EAIgG 114. 35. 37.40]. The contribution of these mechanisms to destruetion of EAIgG anti-D by human monocytes in vilro is not known, bui phagocytosis is not always observed [ I . 2. 19, 23. 32]. Insight into this niechanism is essential, since this system constitutes a model in vitro of the destruction of erythrocytes in vivo in patients with AIHA due to non-complement binding IgG autoantibodies. Serologically and immunochemically such antibodies closely resemble rhesus antibodies and regularly show rhesus specificity [8]. In the present paper the resulls of studies on the mechanism of destruclion of anti-Dscnsitized erythrocytes by human monocyles in vitro are described.
MATERIAL AND METHODS Donors. Venoui blood was collected rrom heallhy donors. .Sera. Fclol culf .scrum (FCS) was obtained from CJihco Biocult. Olusgow. Scoitund, Anii-D sera were ohtuincd from donors who had been immunized against the Rh(D) untigen hy repeated intravenous injections with OR,r erythrocytes. The tiire of ihesc anir-D alloantisera in the indirect aniiglohulin lest varied from 1:2000 to I:I6.(KX> when lestetl with rubbit amihuman globulin serum. Agents. Cyloctiul;isiii B was a product of ICI, Aldcrlcy Park. Cheshire. England, It was dissolved in dimethylsulphoxide (DMSOl to u cuiicenlration ol' 3 mg ml. This solution was further diluted in phosphaicbutTered saline (PBS) lo a conccniraiion of cytochalasin B of SO ^g ml (concentration of DMSO. I" „} and stored at T-4 C, Colchicine was purchased from Mcrcl. AG. Darmstadt, West Germany. Hydrocortisone sodium succinnte wa.s u gifl from Ortfunon, Oss. The Netherlands, Preparation n/ monoiyits. Mononuciear cells were prepared by spinning peripheral blood o%er FicollIsopaque (density 1.077 g,'cm* at 25 C), as described by Loos el at. |24], The resulting intcrfucc layer, whicb contained the mononuciear cells, was washed three limes in minimtil essential medium (MFM) (Gibcu. Grand Islimd. N,Y., USA) supplemented wiih 10",, (V V) FCS. Hcnccfurlh. this medium will be called MEM 10°, FCS. After being washed, the mononuclcar cells were resufpended in MEM 20". FCS to a fmal concentration of 10* celb;ml. This suspension wa* layered on plastic Petri dishes lOptiUix. Falcon Plastics. Oxnard. Calif,, USAt. 2.5 ml per dish, Afler incubalion Ibr 11 h al 37 C, ilic plutcs were vigorously washed to remove the non-adherent cells. The adherent cells were
carefully scraped olt wnli a piece ofsiliconc rubber and resuspended In M I M 10",. FCS. Finally, the cells were washed once, and the cell coneenlralion was adjusted as indicated in Results, The average percentage of monocytes in the adherent cell preparations was 71 I.Tp (mean • SD of twenty donors), as judged by morphology and si/e distribution |25|, Frvf.araiion. labelling and.n-nsitiration o/ eryihrocyte A 5",, suspension of O. rhesus positive (type R^Rj) erythrocyies from freshly drawn heparini/.cd venous blood was prepared in a storage medium containing 2',. (w/vl BSA (bovine serum albumin). The ervihrocyte suspensions were used for 1-2 weeks. Before use the cr>tbrocytes were washed three times in PBS and resuspended in MEM 10"„ FCS to a conccntraiion of 2 IO»;ml. Labelling of erythrocytes. For labelling, equal volumes of erythrocyie suspensions (W/ml) and an Na^^'CrOi solution (Radiochemical Centre. Amersham. England; specitic aaivity 50-400 mCi/mg Cr) were mixed and incubated for 1 2 h at 37 C. The ery ihrocytes were ihen washed three times in MEM 10" „ FCS and were subseiiuenily resuspended to a concentration of I -32 • 10* cells,'ml. Sensitizatiiin itf eryihrocytes. Erythrocytes were sensitized with anti-D tiy mixing ihe erythrocyte suspension 12- 10* mil with an equal volume of undiluted anii-D serum. Subsequently, the eryihrocyte suspension was incubated witb " C r and trealed as described above under 'Labelling of erjihroeyies". Cytutaxicity assay. The cytotoxicity assay was performed as described by Zeijlemaker et al. [421. Briefly. SOij.1 of monocyic suspension (IO"/mll, 50 |/l of anti-DsensitiMd eryihrocytes (l-33> lO"/ml) and 50 \L\ of MFM I0"o FCS were added to wells of round-bottomed microtitre plates. Afler incubation for 16 b at .17 C the plates were centrifuged al 500 g for 10 min at room temperature. A IOO fil sample was taken from the supernatant of each well to determine the percentage of *'Cr release, Specitic cytotoxicily or specific lysis was defined as: E-S
lotal cpm m which £is experimental *'Cr release (monocytes wiih EAIgG anti-D(, S is spontaneous " C r release (monovytes with non-sensiti/ed erythrocytes) and total cpm (counts per ininuti') is total radioactivity per 50 jil erythrocyie suspension. Fach experimeni was performed in triplicate. From tlie percentage of specitic cytotoxicity the absolute number of erythrocytes lysed was calculated by multiplying this percentage by Ihe number of eryihrocyies per well Colchicine and hydrocortisone were dissolved in MFM \Q'\ FCS and added directly lo the wells. Cytochalasin B (50iig/ml in PBS • ! " „ DMSO) was diluted III MEM I O \ r e s and added to the wells to a tinal concentration of ( ixg,ml. The corresponding concentration of DMSO (0.02".. v v» had no influence on cyioioxicity. Fhagoeytosis assay. Phagocytosis was determined from tbe same wells in which lysis ("Cr release) was measured. All bul 25 [xl of the supernatant was carefully removed. Next, 100 [xl distilled water were added
Mechanism of Monocyte Cytotoxic Activity to the wells, and the cell mixture was stirred and incubated for 10 min ai room lemperaiure. Thereafier, the plates were centrifuged. and iOO [j.1 of the supernatant were removed and counted Cor radioactivity. The cell pellets were dissolved in 10% saponine and counted separately Tor radioactivity. The counts of ihe pellet Traction were corrected for ihe 25 [i.1 of ihe "waler" supernatant that remained in the pellet to yield ihe nel cpm of ihe pellet. Phagocytosis was defined as: net cpm pellet — X 100% total cpm Specific phagocytosis was defined as percentage phagocytosis of EAlgG anti-D minus percentage phagocytosis of non-sensitized erythrocytes. From ihe percentage of specific phagocytosis ihe absolute number of erythrocytes ingested was calculated by multiplying ihis percentage by the number of erythrocytes per well. Adherence assay. To determine the number of monocyies binding sensitized erythrocytes. IOO [il of monocyte suspension (2.5 v 10*/ml) were mixed with 50 [xl of a suspension of anti-D-sensitized erythrocyles (2.5-lOx lO'/ml) and 150 |j.l of medium (MEM 10% FCS). The whole was mixed and centrifuged at 150 ^ for 10 min al room temperature. The pellet was carefully resuspended and incubated for I min at room temperature wiih 25IJL1 of a solution containing acrldine orange (4 jAg/mi). The perceniage of roselte-forming monocytes (those that had bound three or more eryihrocytes) was determined with fluoresceni microscopy. Roselte formation with non-sensilized erythrocytes was 0-3%. Release of lysosomal enzymes by manocyles. Monocytes (2x 10') were preincubated for 10 min at 37'C (with or without additions, as indicated). Nexi. either anti-D-sensitized or non-sensitized erythrocyles (4 x 10") were added, and the mixture (volume^ 1.0 ml) was incubated for 60 min at 37'C. Samples of 450 fil were laken al /=Oand /=60 min and centrifuged; the supernatant was collected and Immediately placed on ice. In these supernatants the activities of ihe lysosomal enzymes lysozyme and p-gluciironidase and of the cytoplasmic marker enzyme lactate dehydrogenase (LDH) were determined as described by Goldstein er al. [10]. LDH release was used as an indicator of cell viability.
To determine Ihe lotal activity of these enzymes in the monocytes. the cells were exposed lo 0.2"n (v/v) Triion-X-IOO. The percentage specilic release of these enzymes was calculated by subtracting the percentage release from monocytes incubated wiih non-sensilized erythrocytes from the percentage release from monocytes incubated wiih sensitized erythrocytes. Srarislics. Correlations between measurements were assessed by the rank correlation meihod of Spearman [36].
RESULTS The relation between cytotoxic activity and percentage of monocytes in human mononuclear leucocyte .suspensions The monocyte content and cytoloxic activity of plastic-adherent, non-adherent and ironcarbonyl-treated (for method see Ref. 42) mononuclear leucocyte suspensions arc shown in Table I. It is evident that depletion of monocytes results in an almost complete loss of cytotoxic activity of the mononuclear cells towards EAIgG anti-D. Contribution of lysis and phagocytosis to the destruction of EAlgG anti-D by monocytes The relative contribution of lysis (specific ''Cr release) and phagocytosis (specific *'Cr uptake by monocytes) to the total number of EAlgG anti-D destroyed by monocytes was markedly influenced by the EAlgG to monocyte ratio (Fig. 1). When 5x10^ monocytes were incubated with increasing numbers of EAlgG, the lysis levelled off at an EAlgG to monocyte ratio of 2:1 to 4:1. In contrast, phagocytosis of EAlgG anti-D. negligible at
TABLE T, Relation between cytotoxic activity and percentage of monocytes Jn mononuclear leucocyte suspensions Lysis of EAlgG anti-D (number lysed •; 10=") Mononuclear leucocytes
Adherent Non-adherent Iron-carbonyl-treated MN
517
Percentage monoeytes
1:1»
20:1*
71.2±4.9t 5.9 ±0.9 5.1 ±1.7
26.3 ±2.2t 3.2 + 1.3 2.8 ±0.7
36.2 ±5.5 5.4 ±1.7 5.6 ±1.3
• EAlgG to mononuclear leucocyte ratio. To 5x 10* mononuciear cells 5x 10* or 10" HAIgG were added. The incubation time was 16 h. t Mean + SEM (/i=6).
518
A. Fleer era!. EAH)C
tm • EAlgG proc«ss«d
BO
«0
20
20
ihsgocylosis
05
1
J 4 8 no. EAlgG pof monocylo
16
33
FIG. I. Lysis and phagocytosis of EAlgG anii-D by human monocytes in vitro. Monocytes (5 > 10*) were incubiited wiih variou.s numbers of HAlgG anti-D (5x 10' to 1.6 • IC), resulting in EAlgG to monocyte raiios of 0.5:1 to 32:1 (abscissa). Ordinaie: number of EAlgG processed (i.e. lysed or phagocyti/edl ( • 10^). Incubation time: 16 h. The data shown are averages {mean ±SEM) of experiments with monocytes from eighl donors. • , Lysis of EAlgG; • . phagocytosis of EAlgG; A. toiiil number of EAlgG processed (lysis i phagocytosis).
FIG. 2. Eftect of cytochalasin B on lysis and phagocytosis of EAlgG anti-D by human monocyies. To 5 - 10' monocyies various numbers of EAlgG were added, resulting in EAlgG to monocyle ratios of 0.5:1 10 32:1 (abscissa). Cytochalasin B was added at zero time. Ordinaie: number of EAlgG processed ( • lO^I. Incubation lime: 16 h. The data presented are averages from experiments with moncyles from three donors. • , Lysis of EAlgG; o. lysis of EAlgG in the presence of cytochulasin B (1 pig/ml); • . phagocytosis of EAlgG; I i, phagocytosis of EAlgG in the presence ofcytochaiasin B (1 jig/ml); A, total number of EAlgG processed (lysis ' phagocytosis) in the absence of cy'ochalasin B.
EAlgG lo monocyte ratios of 8:1 or lower, increased at higher ratios. Effects ofcytochaiasin B on lysis and phagocytosis To determine whether monocyies were capable of lysing EAlgG independently of phagocytosis, experiments were performed with cytochalasin B, an inhibitor of phagocytosis [7, 10, 28, 27]. Fig. 2 shows that cytochalasin B (1 M-g/ml) completely blocked phagocytosis, but enhanced the lysis of EAlgG. Moreover, the ntimber of erythrocytes lysed in the presence of cytochalasin B was clearly higher ihan the total number processed in the absence of this agent (the sum of lysis and phagocytosis). This indicates that the enhancement of lysis by this
agent was not only a result of inliibition of phagocytosis. Cytochalasin B is known to enhance the release of lysosomal enzymes by phagocytic celts [7, 10. I I . 13] and its effect on the extracelltilar lysis of EAlgG suggests that lysosomal enzymes released by the monocytes may be responsible for lysis.
Effects of hydrocoriisone and cotchicine on lysis and phagocytosis To find further support for the hypothesis that lysosomal enzymes released by the monocytes are responsible for lysis of erythrocytes.
Mechanism of Monocyte Cytotoxic Activity experiments were carried out with hydrocortisone and colchicine. These agents have been shown to inhibit lysosomal enzyme release by phagocytic cells [ 12, 15, 33, 41. 43]. Fig. 3 shows that both agents inhibited lysis but had no such effect on phagocytosis.
519
Is binding of EAlgG to monocytes required for the occurrence of lysis?
To investigate the requirement of EATgG binding to monocytes for lysis the following experiment was performed: monocytes were incubated with a mixture of non-labelled anti-Dsensitized and "'Cr-labelled non-sensitized erythrocytes. Table III shows that monocytes Relation of the release of lysosomal enzymes by selectively lysed the sensitized erythrocytes, monocytes to lysis whereas the non-sensitized erythrocytes were left unaffected. In a second type of experiment, To obtain quantitative data on the relation monocytes were incubated with increasing between the release of lysosomal enzymes and numbers of non-labelled anti-D-sensitized erylysis of EAlgG by monocytes, these processes throcytes. To these mixtures a small number of were studied in parallel with monocytes of eleven '••Cr-Iabelled EAlgG anti-D C'Cr-EAlgG) were donors. The results are shown in Table II. added. Table III shows that lysis of "^'Cr-EAIgG A highly significant correlation was found decreased when the number of non-labelled between the release of lysozyme and lysis EAlgG per monocyte increased. When mono(r = 0.834; 0.001 > P>0.0005). The correlation cytes and non-labelled EAlgG were centrifuged between release of P-glucuronidase and lysis (5 min at 100 g) before the addition of "Crwas less marked, but still significant (r = 0.620; EAlgG (to establish binding of non-labelled 0,025 > P > 0.01). Table 11 also shows that the EAlgG to the monocytes), this decrease was efFects of cytochalasin B, hydrocortisone and even more marked. Supernatants of mixtures colchicine on the release of lysosomal enzymes of monocytes and EAlgG anti-D (harvested corresponded to their effects on lysis.
PHAGOCYTOSiS
CYTOTOXIC LYSIS EAlgG processed (x10^) 60
€05
40
20 HC1
16 32 1 2 no. EAlgG per monocyte
4
16
32
FIG. 3. EfTccts of hydrocortisone and colchicine on lysis and phagocytosis of EAlgG anti-D by human monocytes. 5x 10' monocyies were incubated with various numbers of EAlgG anti-D, resulting in EAlgG to monocyte ratios of 1:1 to 32:1 (abscissa). Colchicine and hydrocoriisone were added at zero time (final concentrations of I and 5 mM). Ordinate: number of EAlgG processed (x 10*). Incubation lime: 16 h. Data are averages from experimenis with monocytes from three donors. Lcfl: Lysis: A, Control; • , hydrocortisone (! mM); • , hydrocoriisone (5 mM); O, colchicine (t nm); LJ, colchicine (5 niM). Right: Phagocytosis: Symbols the same as with lysis.
520
A. Fleer et at. TABLE II. Lysosomat enzyme release by monocytes: relation lo lysis of EAlgG by monocytes*
Lysosomal enzyme release ("„) Addition Lysozyme Nil(n-ll) I fig/ml cylochalasin B in -6) 5 niM hydrocoriisone (« = 5) 5 mM colchicine (« —5)
fl-Glucuronidase
10.4 r 1.8t
3.8-0.6
18.1,; 4.1 (/'
