Selection of the Delayed Hypersensitivity T Effector and T Suppressor Cell Response by Antigen-Presenting Macrophages

Immunobiol., vol. 168, pp. 246-259 (1984)

Department of Experimental Dermatology, University of Munster, Munster, FRG

Selection of the Delayed Hypersensitivity T Effector and T Suppressor Cell Response by Antigen-Presenting Macrophages ]. KNOP, URSULA MALORNY,

E. MICHELS, and C. SORG

Abstract The T effector lymphocytes of delayed type hypersensitivity reactions (TDH) are regulated by a complex T suppressor (Ts) cell circuit. Induction of T DH cells requires Ia + adherent cells as antigen-presenting cells. Little is known about the antigen presentation for the induction of Ts cells. We describe an experimental model in which TDH and Ts cells are induced separately by different antigen-presenting macrophages grown from bone marrow stem cells. Bone marrow derived macrophages grown in L cell-conditioned medium for various periods and labeled with 2,4-dinitrobenzene sulfonic acid differ in their ability to induce TDH and Ts cells in vitro. The functional activity of the two T subpopulations was assessed in vivo by epicutaneous challenge or sensitization with 2,4-dinitrofluorobenzene of mice receiving the in vitro educated cells. Ear swelling or suppression of swelling was recorded. It could be shown that 5-7 day bone marrow-derived DNP-labeled macrophages preferentially induced Thy 1+ Lyt 1+ antigen-specific TDH cells; 7-10 day old antigen-presenting bone marrow-derived macrophages induced preferentially Thy 1+ Lyt2+ antigen specific Ts cells. Characterization of various phenotypic markers revealed different surface antigen expression and functional differences such as MIF responsiveness or transglutaminase activity on the two macrophage populations. These data support the concept that activation of the Ts regulatory circuit may require antigen presentation by specialized antigen presenting cells, characterized by certain surface and functional markers and different from those inducing preferentially T DH cells.

Introduction The delayed type hypersensitivity (DH) reaction induced and elicited by various contact sensitizing agents such as 2,4-dinitrofluorobenzene (DNFB) or picrylchloride (PCL) is regulated by cellular interactions between various T cell subpopulations and antigen-presenting cells (APC). The clonal expansion and the functional expression of the T cell subset (TDH cell) that mediates the DH reactions in these models is controlled by a complex T suppressor (T5) cell circuit (reviewed in 1-3). There is considerable evidence that Ia+ adherent cells such as macrophages function as accessory cells in several immune responses including delayed type hypersensitivity reactions (4-7). Little is known about the primary events leading the induction of the T5 cell circuit of the DH reactions. In the following presentation we will briefly discuss the role of APCs in the

In vitro induction of T effector and T suppressor cells . 247

induction of the T5 regulatory circuit in contact sensitization and summarize our own data pertinent to this question.

The

r. regulatory circuit of contact sensitization

Administration of a contact allergen or other antigens eliciting DH reactions by a route which avoids sensitization such as oral feeding, intravenous injection of allergen (9-11), removal of the local sensitizing depot of allergen (12), antigen overload (13) or haptenized lymphoid cells (14) results in tolerance partially or completely mediated by T5 cells (15-20). In contact allergy and related models the following T5 types have been recognized: a) an afferent acting T5 cell (T5 aff) which suppresses the development of the TDH cell (21-23), b) an efferent-acting T5 cell suppre.psing the function of the already established TDH cell (24-26) and c) a T53' T~lacceptor (T5 acc) or auxiliary Ts (Ts aux) cell which appears to be the final T suppressor effector cell (27-29). The sequence of induction of the Ts subtypes may be different depending on the allergen used. These Ts subtypes appear to interact by a pathway of alternating self-idiotype reactions mediated by Ts suppressor factors which are partially MHC (1-]) restricted (30-32). A summary of the properties of the various Ts subtypes and some of their purified suppressor factors has been published (33).

r. activation and modulation There is a general agreement that the activation of TDH requires at least two signals. Signal 1 is provided by antigen in association with Ia antigen, usually exposed on the surface of APCs. Signal 2 appears to be interleukin 2 released from T helper cells through the action of interleukin 1 provided by the accessory cell. The signals required for T5 activation are far from clear. Induction of T5 is obtained by presenting the antigens by routes which usually do not allow sensitization (see above). This suggests that Ts-specific signals differ from those required for TDH activation (for discussion see reference 34). It should be mentioned, however, that presentation of antigen in association with Ia may efficiently induce tolerance (35) and that growth of T5 (36) or Lyt2+ cells is supported by IL2 (37). The T5 circuit can be modulated by a recently described contra suppressor cell circuit (38, 39) or by signals given by unspecific immune modulators. Tolerogenic signals can be converted into immunogenic ones by immune modifying agents such as ConA, amphotericinB (41), dextran (42) and poly-AU (43). Poly-AU (44) and lipopolysaccharide (45) have both been reported to prevent the development of suppressor T cells. We have found that tolerance induction by 2,4-dinitrobenzene sulfonic acid (DNBS03) or by antigen overload using 2,4-dinitrofluorobenzene (DNFB) was partially or completely prevented by prior injection of Corynebacterium parvum (c. parvum) (46, 47). A similar effect could be observed by using serum obtained from C. parvum-treated animals (Fig.1); the T5

248 .

J. KNOP, URSULA MALORNY, E. MICHELS, and C. SORG TS donor treated (in vivo)

% suppress i on

Ts-type transferred

20

60

40

I

I

TSaff C.p.

~s

C.p. serum

~s

C.p. serum + anti-IFN IFN

I

"

~s

103U

I

TSeff IFN

3 5x10 U

Js

Fig. 1. Inhibition of the T,-aff and T,_df response by C. parvum (c. p.) serum obtained from mice treated 24 hr before with C. parvum and highly purified mouse IFN a,~. T,-aff and T,_o££ were induced in non-treated donors, donors treated 2 days before with C. p. or treated with C. p. serum, C. p. serum plus anti-IFN globuline (polyclonal) or IFNa, ~ within 1 hr after i.v. injection of a tolerogenic dose of DNBSO) (T,_,ff induction) or DNP spleen cells (T,_ff induction). Six days later spleen cells from these mice were transferred to either naive DNFB sensitized animals to assay for afferent and efferent suppression, respectively. The mice were sensitized and ear challenged or ear challenged only with DNFB and ear swelling was measured 24 hr later. The results are expressed as percent suppression of the ear swelling in optimally sensitized animals minus the toxicity control. s = significantly different.

inhibitory activity in the serum could be neutralized by an anti-interferon globuline (Fig. 1, 48). Injection of a low dose of interferon a, ~ sh<;>rtly after Ts induction prevented the development of Ts cells without affecting the T DH response (Fig. 2, 49). In vitro treatment of Ts aff with IFN inhibited its T-cel1 transferred

TDH

TSaff

treated in vitro before trans fer wi th

control buffer IFN

,104U/108cel1s

IFN

,103U/108cel1s

control buffer

ear swel1ing (10- 2 mm) 10

15 I

20

25

I

I

30 I

J ns Ins Is

J

Fig. 2. Selective inhibition of T,_,ff by murine IFNa, ~ in vitro. Lymph node cells containing TDH cells were prepared 4 days after optimal sensitization from sensitized animals, T,-aff containing spleen cells from animals receiving a tolerogenic dose of DNBSO) 6 days before. The cells were incubated with IFNa, ~ or control buffer for 2 hr, thereafter washed three times and injected intravenously into naive recipients which were challenged immediately afterwards (assay for TDH cells) or sensitized twice (day 0 or 1) and ear challenged (day 4 assay for T s- aff cells). Ear swelling was measured 24 hr later. ns = nonsignificant, s = significant.

In vitro induction of T effector and T suppressor cells . 249

suppressive activity assessed by transfer in vivo whereby the TDH cell was not affected by IFN in vitro (Fig. 2, 49, KNOP et aI., submitted). These observations provide a link between immune stimulation, inhibition of the Ts cell circuit and IFN.

The role of antigen-presenting macrophages for the induction of the pathway

'L

Activation of T DH cells is efficiently accomplished by specialized antigenpresenting cells, such as dendritic (50) and Langerhans' cells (51). In many in vitro systems antigen-specific T helper cells are generated in the presence of adherent cells while removal of adherent cells results in the induction of Ts cells (52-54). Similarly, application of contact allergen to animals devoid of intact antigen-presenting Langerhans cells or sensitization of animals treated before with antibody specific for gene products of the IA subregion not only prevents specific immunization but favours the induction of antigen-specific Ts cells (55-59). These data suggest that bypassing IA or IE subregion restricted antigen presentation will activate the Ts circuit; they do not exclude some form of antigen presentation necessary for the induction of Ts cells. Indeed, there are some reports which suggest that specialized APCs are required for the induction of at least one subset of T suppressor cells, the Ts3 or Ts effector cell (60-62). This APC has been identified as an I-r I-A - cell (62,63). Cooperation between APCs and various T lymphocyte subsets involved in regulatory circuits of DH is not restricted to antigen presentation in association with I-region encoded antigens but is modulated by the functional and secretory state of the presenting cell (64-66). Secretory products released by macrophages after contact with immune stimulants or antigens, such as interleukin 1 (67,68), interferon (69, 70) and prostaglandins (71, 72) have been shown to affect lymphocyte proliferation and differentiation. Interferon can be produced by macrophages activated by C. parvum (73); its release by an APC could unspecifically prevent the induction of Ts cell, thus allowing the induction of an effective T DH response. Growth factors or growth inhibitory factors may be - in addition to appropriate antigen presentation associated with MHC-restricted antigen - important signals for the activation or suppression of TDH or Ts cells.

Induction of TDH and Ts cells in vitro by haptenized bone marrow-derived macrophages

In order to study the role of various macrophage populations as APCs for the induction of T DH and Ts cells in contact sensitivity we developed an in vitro system which allows to induce TDH and Ts lymphocytes, using cultured bone marrow-derived macrophages (BM-MA) as APCs (KNOP et aI., submitted). The macrophages used in this study were grown from bone marrow cells in a liquid culture system, using 20 % L cell-conditioned medium. Details of the culture techniques and characterization of mac-

250 . J. KNOP, URSULA MALORNY, E. MICHELS, and C. SORG

rophages grown from bone marrow stem cells are described elsewhere (74). After 5-7 days 95-99 % of the cells show the characteristics of macrophages. In the present study BM-MA grown for 5,7 or 10 days in L cellconditioned medium were plated into 20 mm plastic tissue culture dishes and allowed to adhere. The nonadherent cells were washed away and the adherent cells pulse-labeled with DNBS03. After washing, spleen lymphocytes without adherent cells were added to the DNP-Iabeled BM-MA. After culture for three days the lymphocytes were recovered and injected intravenously into BALB/c mice which were challenged with DNFB within 1 hour after cell transfer (T effector cell test) or sensitized twice with 0.5 % DNFB after cell transfer (suppressor cell test). BM-MA obtained from bone marrow cultures grown for 5 or 7 days in L cell-conditioned medium (BM-MA d5 or d7) were able to induce a T effector cell response in vitro, which could be detected after transfer into nonsensitized animals which were subsequently challenged (Fig. 3). BMMA d10 did not induce T effector cells (Fig. 3). BM-MA d10 and d7, however, induce suppressor cells (Fig. 4). No suppressor cell response was observed using lymphocytes from co-cultures with 5 day old BM-MA. To exclude differences in antigen binding by 10 and 5 day old adherent BM-MA, they were pulse-labeled with 3H-DNFB under similar conditions as used for DNBS0 3 labeling. BM-MA on day 5 bound slightly less DNFB (1.18 f-tg DNFB/107 cells) than BM-MA on day 10 (1.52 f-tg DNFB/107 cells), an insignificant difference. To characterize the in vitro induced T effector lymphocytes DNP-BM-MA d5 were used as APCs. Treatment of the «educated» lymphocytes with anti-Thy 1.2 or Lyt 1.2 plus complement before transfer into naive recipients and DNFB challenge completely destroyed the ability to transfer contact sensitivity. Anti-Lyt2.2 serum plus complement or complement alone had no effect (Fig. 5). The TDH cell was BM-~1a.

of culture

4

ear swelling (10- 2 mm)

day DNP-labeled

6

8

10

+ +

+

10

4

I -_ _ _ _ _ _ _ _ _ _ _- - -JI s

Is

+

Fig. 3. Induction of TDH cells by DNP-BM-MA. BM-MA cultured for various periods (days) in L cell-conditioned medium were allowed to adhere in tissue culture plates and after pulselabeling with DNBS0 3 were incubated with spleen lymphocytes for 3 days. The lymphocytes were harvested and injected i.v. into naive recipients which were challenged with DNFB 1 hr later. s = significant.

In vitro induction of T effector and T suppressor cells . 251

BM-MA

%suppression

day of

culture

DNP-label.

20

40

60

I

5

+

7

+

Is

10

+

1---_ _ _ _......1 s

10

Fig. 4. Induction of suppressor cells by DNP-BM-MA. Spleen lymphocytes obtained from coculture with DNP-Iabeled BM-MA, cultured for various periods (days) in the presence of L cell-conditioned medium were injected i.v. into naive recipients which were sensitized afterwards with 0.5 % DNFB and challenged on day 4. The results are presented as percent suppression of the ear swelling in optimal sensitized animals (positive control minus toxicity control). s = significant.

antigen-specific (Fig. 5). The suppressor cells induced by co-culture with DNP-BM-MA dl0 were Thy 1+, Lyt2+, and also antigen-specific in their effector function (Fig. 6). Functionally the Ts induced in vitro could be characterized as afferent acting cells, since they caused maximal suppression if injected prior to sensitization. Injection into sensitized animals caused insignificant suppression.

Induction of TDH and

t

cells by DNP-BM-MA in vivo

DNP-BM-MA d5 or dl0 cultured for 3 days after antigen pulsing induced neither contact sensitivity nor tolerance when injected i.v. Into cha llenge of' treatment of TDH before transfer

ear swelling (10- 2 mm)

recipients with

DNFB

6

4 I

~

I

______________

~I

8

10

s

pcr C'

DNFB

r-______________

~Is

anti-Thy 1.2+C' "

-Lytl.2+C'

"

- Lyt 2. 2+C '

Is

Fig. 5. Characterization of the TDH cell induced by 5 day old DNP-BM-MA. For technical details see legend Figure 3. The recipients of the in vitro educated TDH were challenged with DNFB or 1 % picrylchloride (PCL). s = significant.

252 .

J.

KNOP, URSULA MALORNY, E. MICHELS, and C. SORG

treatment of Ts before transfer

sensitization and challenge with

C'

%suppression

20

40

DNFB

60

80

1s

anti-Thy 1.2+C'

" -Lyt 1.2+C'

1s

" -Lyt 2.2+C'

PCI Fig. 6. Characterization of the suppressor cell induced by 10 day old DNP-BM-MA. The recipients of the in vitro educated Ts cells were sensitized with DNFB (0.5 %) or PCL (7 %) after the cell transfer and challenged on day 4 with DNFB or PCL. The results are presented as percent suppression (see legend Figure 4). s = significant.

sensitized or nonsensititzed animal.s. In contrast, BM-MA used directly after recovery from conditioned medium cultures without prior adherence and pulsed with DNFB were effective when injected subcutaneously. DNPBM-MA dS were able to induce contact sensitivity in the recipients while DNP-BM-MA di~ had significantly less sensitizing capacity (Fig. 7). On the other hand, DNP-BM-MA ds and di~ were both able to induce splenic . Ts cells in vivo (Fig. 8). These Ts cells were able to suppress the TDH cell at the effector stage; they appeared therefore to be efferent acting'Ts cells, in contrast to the Ts subtype induced in vitro which suppressed the afferent limb of the DH response. This may be explained by a rapid induction of Ts eff in vivo by Ts aff acting as inducer cell. In vitro the suppressor circuit may be arrested at the Ts aff stage and Ts eff cannot be demonstrated. BM-Ma. used after culture period (days)

ear swelling (10- 2 rivn) 4

DNFB-l abe 1.

+

8

10

Is

+

10

6

1--_---11 ns

10

Fig. 7. Subcutaneous sensitization by DNP-Iabeled BM-MA cultured for 5 or 10 days. BMMA cultured for 5 or 10 days in L cell-conditioned medium were DNP-Iabeled without prior adherence and 2 X 107 cells per dose were injected on day 0 and 1 subcutaneously. On day 4 ear challenge was performed and ear swelling measured on day 5. ns = not significantly different from controls, sensitized with non-labeled BM-MA, s = significantly different from controls.

In vitro induction of T effector and T suppressor cells . 253

BM-Ma day

DNFB-label.

5

+

10

+

trea tment of Ts before transfer

+

C'

+

anti-Thy 1.2+C'

% supression

20

40

60

Fig. 8. Induction of T, cells by DNP-BM-MA d5 and 10 in vivo. BM-MA cultured for 5 or 10 days in L cell-conditioned medium were DNP-Iabeled without prior adherence and injected i.v. (5 X 106 cells/mouse) on day OJ spleen cells from these mice were transferred on day 6 to sensitized recipients 1 day before ear challenge. s = significantly different from non-labeled control.

Characterization of BM-MA d5 and dlO Mouse bone marrow cells differentiate in culture in the presence of L cell-conditioned medium to macrophages. They go through a phase of intensive proliferation (74), then differentiate into macro phages and are fully mature after 5-6 days on the basis of phenotypic markers, such as cell morphology, nonspecific esterase, phagocytosis of latex beads, Fc receptor expression and Fc receptor-mediated phagocytosis (74). The results of experiments in which proliferation, release of plasminogen activator, expression of trans glutaminase, random motility and response to a standard preparation of purified macrophage migration inhibitory factor (MIF) were recorded daily are shown in Figure 9 (MICHELS et al., submitted for publication). As demonstrated in Figure 9, during the course of maturation the various phenotypic markers recorded show remarkable changes. After proliferation is subsiding, the expression of trans glutaminase activity is increasing from about 10 % in 5 day BM-MA to almost 100 % in 10 day BM-MA. Plasminogen activator production was transiently expressed at day 4 and 12. Random motility of cells determined as distance of migration from an agarose droplet was high at the beginning of culture, but steadily declined thereafter, being low in 10 day BM-MA. The response to MIF obtained from supernates of Con A-stimulated spleen cells and purified by Sephadex chromatography and isoelectric focussing was high in the 5-8 day BM-MA, whereas BM-MA d10 did not react with MIF at all (Fig. 9). These data together with others not shown here, support the conclusion that the MIF-sensitive macrophage phenotype has finished cycling and has arrived in G j • With no further mitogenic signal added, the cell progresses to mid

254 .

J. KNOP, URSULA MALORNY, E. MICHELS, and C. SORG ,.... (')

'0 ~

x

. a. 10

(A)

u

'5

±

5

(')

~ 100

~

ui

8. ell :l

1.0,....

50

c 05 .....

.a.

~

(C)

2.

50~ 30

~ c

2

4

6

8

( days)

10

12

14

Fig. 9. Maturation kinetics of bone marrow-derived macrophages. 3H-Thymidine tH-TdR) incorporation (. -+) of a bone marrow liquid culture (A). Kinetics of plasminogen activator (PA) secretion (e-e) and intracellular transglutaminase (TGase) expression (0-0) (B). Relative cell motility, determined as distance of migration (0-0) from an agarose droplet and MIF response (.-.), expressed as diluting factor that causes 30 % migration inhibition (C).

G h where high amounts of trans glutaminase are expressed. MIF responsiveness may be induced again by a mitogenic signal, i.e. colony stimulating factor and passage through the cell cycle. The TDwinducing BM-MA seems to be a MIF-responsive cell, whereas the Ts-inducing BM-MA rather is a MIF-unresponsive macrophage. In this case, induction of the MIF-responsive state in otherwise MIF-unresponsive macrophage phenotype by a mitogenic signal should transform the Ts-inducing BM-MA into a T Dw inducing APC. These experiments are in progress. The two bone marrow populations studied in our system also showed differences in the expression of certain membrane antigens, identified by monoclonal antibodies. The monoclonal antibodies have been obtained by immunization against bone marrow derived or normal peritoneal macrophages (MALORNY et aI., submitted). An increase in the number of positive cells judged by cell sorter analysis was found in the day 10 population of BM-MA with the monoclonal antibodies BM 8 and BM 11. No difference between the two populations was found with NP 7 (Table 1). It should be mentioned that these three monoclonal antibodies do not recognize granulocytes, mast cells fibroblasts, spleen lymphocytes and thymocytes or platelets. Using a rat anti-mouse Ia monoclonal antibody (Hybrotech Inc., San Diego, CA), only 15-16 % of the cells in both BM-

In vitro induction of T effector and T suppressor cells . 255 Table 1. Antigen expression by bone marrow-derived macrophages

BM 8 BM 11 NP 7 Ia

BM-M«I> d 5 % positive cells

BM-M«I> d 10

76

92 92 78 16

65 73 15

- Test indo immunofluorescence - Data by EPICS V Evaluation of 30.000 cells per sample

MA populations expressed Ia antigens with no difference in the two populations. These observations, however, require further substantiation, using other Ia antisera, particularly in the light of results obtained by LEE and WONG (75), who found in parallel with the occurrence of la-positive BM-MA a maximal antigen-presenting activity of these cells during the exponential phase of growth; Ia expression and antigen-presenting activity declined as the cells entered the stationary phase at day 8.

Conclusions Using bone marrow-derived macrophages of different culture age, two separate populations of APCs can be recognized which preferentially in duce T DH or Ts cells. A third population appears to be able to induce both types of T subpopulations. BM-MA proceeding from growth to the stationary phase are progressively loosing antigen-presenting activity for the induction of T DH , gaining antigen-presenting activity for Ts cells. Certain phenotypic characteristics are associated with the T DW and Ts-inducing BM-MA population: The TDwinducing BM-MA is a MIF-responsive population, having finished cell cycle and arrived in G j • Certain antigenic markers recognized by the monoclonal antibodies BM 8 and NP 7 are less expressed on this BM-MA population than on those preferentially inducing Ts. This latter population is further characterized by MIF unresponsiveness and high trans glutaminase expression. Macrophages are a heterogeneous population of cells which differ in the inflammatory effector function, the expression of Ia antigen and antigenpresenting function and, as recently described, in the ability to select specific T cell functions (76, 77). Our data would support the concept that activation of the Ts regulatory circuit may require antigen presentation by specialized APCs characterized by certain surface and functional characteristics different from those which act as APCs for T DH cells. Selective Ts inducing ability may not be a fixed function of certain macrophage type but

256 .

J. KNOP, URSULA MALORNY, F. MICHELS, and C. SORG

rather a transient characteristic. Similarly as has been described above for MIF-responsiveness, a Ts-inducing APC may be converted to a T DW inducing macrophage by an appropriate signal. The phenotypic change would not only involve the expression of surface markers but also the release of mediators such as IL 1, interferon or prostaglandins, which will preferentially activate or suppress certain T cell functions. Our system described above would allow us to solve some of these questions. Acknowledgement We wish to thank BRIGITTE BROKMEIER for excellent technical assistance and BRUNHILDE SCHEIBEL for typing the manuscript. This work was supported by grant SFB 104/C5 from the Deutsche Forschungsgemeinschaft.

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l KNOP, M. D., Ph. D., Department of Dermatology, University of Munster, VonEsmarch-Str. 56, D-4400 Munster, FRG