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Immunomodulatory effect of synthetic peptides corresponding to sequences within the CH2 and CH3 domain of human IgG1
Mobcular Immunoiogy,Vol. 25, No. I I, pp. 1183-l188, 1988 Printed in Great Britain.
0161.5890!88$3.00f0.00 Pergamon Press plc
IMMUNOMOD~LATORY EFFECT OF SYNTHETIC PEPTIDES CORRESFONDIN~ TO SEQUENCES WITHIN THE CH2 AND CH3 DOMAIN OF HUMAN IgGl GABRIELLA SARMAY,* KRISZTINA REGULY,* ROBERT SZICIETI,~ EVA KLEIN,? DENIS R. STANWORTH: and JANOS GERGELY* *Department of Immunology, L. E6tv8s University, G6d. Hungary: TDepartment of Tumor Karolinska
Institutet,
Stockholm,
Sweden; and $Department U.K.
of Immunology.
University
Biology, of Birmingham.
Fc region of IgG is known to be the source of small peptides possessing immunomodulatory function. Results are summarized showing the effect of synthetic peptides composed of surface exposed residues of Cy2 or Cy3 domains on different steps of human B lymphocyte activation
Abstract-The
cycle. Both the CH2 (289Thr-3OIArg) and CH3 (4~7Tyr~~6Arg) peptides as well as the whole Fe fragment enhanced the IgM synthesis of PWM or PMA + CaI activated lymphocytes. This effect was exerted at the early phase of B cell activation. The incubation of separated resting B cells with Fc fragments or CH2 peptides resulted in increase of cell volume and in expression of HLA-DR antigen. On the other hand, LIF production was induced both by CH2 and CH3 pcptides. It was also shown that Fe peptides induce IL-1 release from monocytes. The results suggest that the CHZ and CH3 domain peptides exert their effect partly directly, by activating resting B cells, rendering the cells more susceptible to other stimuli: and moreover. by enhancing the humoral response by triggering the release of IL-I.
INTRODUCTION
Immunoglobulins secreted by activated B cells have a multipotential role in regulating the immune response. Antigen-~omplexed IgG molecules are able to bind to specific receptors (FcR)* on the surface of different cell types, thereby in~uencing functions such as phagocytosis of macrophages, antibody dependent cellular cytotoxicity (ADCC) of K cells and regulation of Ig synthesis by B cells. Both the enhancement and the inhibition of antibody production induced by IgG molecules or its fragments has been described (Sinclair and Panoskaltsis, 1987). Crossbinding of sIg and FcR on the surface of the same B cell results in the inhibition of Ig synthesis (Kolsch et al., 1980, Uher and Dickler, 1986). On the other hand, Fc fragment of IgG molecule and a peptide (~23) released from the Cy3 domain by surface enzymes of macrophages have been shown to stimulate the antibody productjon of mice spleen ceils (Morgan et al., 1983, Hobbs er al., 1985). Recent findings suggest that various small mol. wt peptides possessing immunomodulatory function _____~_. --__-_. *List of abbreviations:
ADCC, antibody dependent cellular cytotoxicity; Cal, Ca ionophore A23187; FcR, receptors interacting with the Fc portion of IgG; FITC, Auorescein-isothiocyanate; IL-I, interleukin-1; IL-2. interleukin-2; LIF, leukocyte migration inhibitory factor; MHC, main histocompatibiiity complex; PBMC, peripheral blood mononuclear cells; PHA, phytohaemaglutin~n~ PMA, phorbol-12 myristate-13 acetate; PWM, pokeweed mitogen; sIg. surface immunoglobulins. §Correspondence to be addressed to: Gabriella Sarmay, Department of Immunology, L. EEitviis University, Javorka S.U. 14, Gild, 2131 Hungary.
might be released from the Ig molecules in citv. Tuftsin (289Thr--292Arg), one of the best characterized peptides amongst these influences mainly the function of macrophages (Najjar, 1985; Florentin et al., 1986). A Cy 3 domain peptide (345Glu-349Arg) called rigin has also been shown to have a tuftsin-like effect (Veretennikova et al., 1981). More recently, a ~ntapeptide (351 Leu-355Arg) was found to be capable of demonstrating the immunostimulato~ effect of the peptide p23 (Hobbs et al., 1987). A decapeptide representative of a sequence within the CH4 domain of the human IgE molecule has been shown to induce histamine release from mast cells in a manner closely resembling the IgE dependent triggering process (Stanworth, 1982). Since some of these peptides has been shown to be released in a&o as well, one may predict that they are localized on the surface of Ig molecule, where they would be available for enzymatic cleavage. On the basis of their hydrophilicity and accessibility, groupings of certain amino acid residues within the IgG Fc region can be predicted to constitute antigenic epitopes on the surface of the Cy2 and Cy 3 domains (Martinez and Winternitz, 1983). These sites are available for interactions with other cell surface receptors as well as being vulnerable to enzymatic cleavage. Some of these surface exposed hydrophilic patches, like those composed of residues 285-293 in the CH2 or 33 l--346 in the CH3 domains of IgG molecules incorporate sequences corresponding to peptides with known immunoregulatory properties. Taking into consideration these facts, we have planned and synthesized peptides comprising further
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GABRIELLA S~RMAY
et al.
Table I. Location of synthetic peptides on IgGl Fc Peptides
Sequences
CH2 domain: Y48 Y91
(289)Thr-LysPro-Arg-Gl~Gl~Gl~Tyr-AsSer-Thr-Tyr~Arg(3~~) (293)GI~GlbGI*Tyr-Asp%-Thr-TyrArg(3Ol)
CH3 domain: Y75
(407)Tyr-SermLys-LewThr-V&AspLys-Ser-Arg(4l6)
Bold characters stand for the predicted antigenic epitopes of IgGl (Martinez and Winter&z, 1983)
surface exposed sequences of the IgG molecule and tested their effects on different steps of the human lympocyte activation cycle. RESULTS
Synthetic peptides composed of surjtice exposed residues of Cy 2 or Cy 3 domains enhance IgM synthesis by stimulated human B lymphocytes The synthetic peptides studied in the following experiments are listed in Table 1. Peptide Y48 includes the sequence corresponding to tuftsin, while peptide Y91 represents the sequence downstream from the tuftsin. Peptide Y75 contains hydrophilic residues of the CH3 domain and is different from the one studied earlier by others (Hobbs et al., 1987). Peptides Y48, Y91 and an analogue of Y75 have been found previously to inhibit the FcR mediated ADCC (S&may et al., 1984). Some of the results presented here have been published in detail elsewhere (Sdrmay et al., 1988). The influence of the peptides and that of the Fc fragment or the whole IgG molecule on B cell stimulation was studied first using the in vitro cultures of peripheral blood mononuclear cells activated with suboptimal amount of pokeweed mitogen (PWM). The proliferation of these cells was not affected by Fc fragment or Fc peptides, whereas they significantly enhanced IgM synthesis of activated B cells as detected in the supernatants of the cultures by ELISA (Table 2). In contrast, the whole IgG molecule had no effect. The peptides or Fc fragment alone, in the absence of PWM, failed to induce either proliferation or antibody synthesis (data not shown). Both the mitogen and the Fc peptides needed to be present from the initiation of the culture in order to induce an optimal enhancement of IgM secretion, whereas, adding them at the third day had no significant effect.
The response to PWM is macrophage and T cell dependent. Therefore, in further experiments, separated B cells were stimulated directly by phorbol-12myristate- 13-acetate (PMA) plus Ca ionophore A23187 (Car) in the presence and in the absence of peptides, and IgM secretion into the supernatant of the cultures was compared. Fc fragment and Fc peptides induced an enhanced IgM synthesis compared to the samples stimulated with PMA + CaI alone. Fc fragment induced some IgM synthesis in the absence of additional stimuli as well (Fig. 1). Fc peptides induce interleukin-1 (IL-l) monocytes
Fc fragment was previously shown to induce IL-l production by macrophages (Oppenheim et al., 1986). Adherent cells from peripheral blood monocular cells were cultured in the presence of Fc frag100
r
i FC
[il lbl Y48
content of supernatant was measured by ELISA assay. The first columns represent the samples cultured without PMA and CaI, the second columns demonstrate the IgM level in stimulated cultures.
IgM content of the supernatants (ng/ml) YlS Y48 Y91
FC
1st day 3rd day
985 f 305 nt
798 f 30 327 + 30
903 i 30 432 + 50
726% 15 380 F 40
5
1st day 3rd day
610 + 101 nt
482 f 100 272 f IO
449 + 100 326 F 35
390 f 40 2X4 k 80
0.5
1st day 3rd day
465 k 63 “t
318 + 60 231 + 80
340 f 20 25X k 60
324 + 40 205 f 70
Control:
Y75
Fig. 1. Fc peptides and Fc fragment enhance IgM synthesis induced by PMA plus Ca ionophore A23187. Separated B cells were stimulated by 1ng/ml PMA and 0.5pg/ml Ca ionophore in the presence and in the absence of Fc fragment or Fc peptides (10 ,~g/2 x 10S cells) for 8 days. The IgM
Table 2. Fc fragment and synthetic peptides corresponding to sequences withm the CH2 or CH3 domain of IgGl enhance PWM driven IgM synthesis Peptide doses (pglml) 50
release from
244 f 90 ng/ml
Fc fragment or Fc peptides were added either at the initiation OTcultures together with PWM (1st day), or after 3 days stimulation with PWM. The IgM content was measured by ELISA assay on the 9th day. Mean k S.E. of 6 experiments.
Immunomodulatory
effect of synthetic
. . I
. .
4 . :
. . Ic
:
.
.
-+
L
I
I
I
I
I
Fab
Fc
Y48
Y91
Y75
Fig. 2. Fc peptides as well as Fc fragment induce IL-I production by monocytes. Adherent cells were purified from PBMC in 24 well Costar plates by incubation of 5 million cells in 1ml complete medium at 37°C for 2 hr followed by the vigorous washing of the non-adherent population. Fifty pg Fc fragment or peptides were added to the cultures of 1 ml medium, and the samples were incubated at 37°C for 18 hr. The supernatants were harvested and tested in the standard thymocyte-costimulatory assay. The results are expressed as stimulation indices: [‘HI thymidine incorporation in test supernatants divided by that of the control cultures incubated in medium alone.
ment and Fc peptides for 18 hr, and the supernatants of cultures were tested in the standard thymocyte co-stimulatory assay (Fruehauf et al., 1982). Both the Fc fragment and the Fc peptides induced IL-1 synthesis by monocytes. The mean stimulation indices were 2, 1.6 and 2.1 for Fc fragment, Y48 and Y75 respectively. Fab fragment did not induce IL-l production. The amount of IL-l detected in the supernatants of Fc fragment or peptide treated monocytes corresponded to 400U/ml IL-l compared to thymocyte proliferation induced by recombinant IL-l standard (Fig. 2). In the supernatant of PBMC cultured in the presence of peptides only a moderate, insignificant, amount of IL-1 was detected, probably because of the consumption of the synthesized IL-I by the cultured cells. Supernatants of the monocyte depleted cultures had no effect on thymocyte proliferation (data not shown). The Fc peptide induced supernatants were also tested for the presence of interleukin-2 (IL-2). The 48 hr supernatants of PBMC cultured with Fc fragment for Fc peptides did not contain a detectable amount of IL-2 as assayed in the standard IL-2 dependent CTLL proliferation assay (Gillis and Smith, 1977) (data not shown). IL-2 receptor expression has also not been detected on the peptide treated T cells. The effect oj’Fc,fragments and Fcpeptides on the early signs of B cell stimulation B lymphocytes have been shown to undergo some rapid changes after being exposed to different stimuli.
1185
peptides
For instance, they might produce leukocyte migration inhibitory factors (LIF), express a higher amount of MHC class II antigen on their surface and increase in volume (Szigeti, 1985; Kehrl et al., 1985). To test the possibility that Fc fragment and peptides might have a direct effect on B lymphocytes, the resting B subset was separated by Percoll gradient centrifugation, and cultured in the presence of these reagents for 18 hr. The supernatants of these cells were tested for the presence of LIF in a granulocyte migration inhibitory assay. while the resting B lymphocytes from the same cultures were assayed for the of HLA-DR antigen by indirect expression immunofluorescence. Both Fc fragment and Fc peptides induced dose dependently LIF production by resting B, but not by T lymphocytes (Fig. 3) nor by already activated B cells (data not shown). The amount of LIF induced by the peptides or Fc fragment was comparable to that measured in the anti-IgM stimulated B cell cultures. T lymphocytes produced LIF when stimulated with PHA (Fig. 3). The expression of HLA-DR antigen on resting B cells was significantly enhanced as a result of culturing them overnight with Fc fragment or Y48 and Y91 peptides compared to control samples, whereas peptide Y75, the CH3 domain peptide had no effect. The volume of the positive cells also increased in the former cases. Figure 4 demonstrates a representative experiment obtained with Fc fragment and peptide Y48. Taken together these findings indicate that Fc fragment and synthetic peptides corresponding to surface exposed sequences within the CH2 or CH3 domain are able to induce the appearance of certain early signs of activation on resting B lymphocytes, although peptides comprising sequences from the CH2 and CH3 domain differed in respect to their effect on HLA-DR expression.
c
c
Fob
FC
Y49
Y91
Y75
PHA Antim
Fig. 3. LIF production by separated resting B (N) and T (0) lymphocytes cultured with Fc fragment (30 pg/ml), Fc peptides (100 pg/ml), anti-IgM (30 pg/ml) and PHA (2 pg/ml) respectively, overnight. Supernatants of the samples were tested by indirect leukocyte migration inhibitory assay. Control samples were treated with medium only. Mean + SE. of 8 experiments.
GABRIELLA SARMAY et al.
1186
Channel
number
Fig. 4. Increase of the cell volume and HLA-DR expression on resting B lymphocytes cultured with Fc fragment or the CH2 domain peptide Y48. Control samples were cultured in medium only. The cells were treated with monoclonal antibody specific for HLA-DR. then with FITC-conjugated anti-mouse Ig. The samples were analysed by FACS.
DISCUSSION
Low mol. wt peptides from various protein molecules might be released by surface enzymes of different cell types. Such enzyme activity has been detected on macrophages, activated T cells and lymphoblastoid B cell lines (Morgan et al., 1982; Erdei et al., 1984; Ramos et al., 1985). The cleavage of the IgG molecule following its binding to FcR on macrophages was demonstrated earlier (Morgan er al., 1983). Peptides corresponding to surface exposed sequences of immunoglobulins has been shown to possess an immunomodulatory effect (Martinez and Winternitz, 1985). Synthetic peptides comprising sequences within the CH2 and CH3 domain of IgGl were previously found to inhibit ADCC suggesting, although only indirectly, that they might bind to the receptors specific for the IgG Fc (Sarmay et ul.. 1984). On the other hand, the possibility that the Fc peptides act through binding to other structures on the cell surface cannot be excluded. FcR play an important role in the regulation of antibody production. Depending on the interactions of ligands with FcR or antigen receptors on the surface of B lymphocytes, the antibody synthesis can be up- or down-regulated (Sinclair and Panoskaltsis, 1987). Peptides originating from the Fc part of IgG molecules and possibly released in aivo as well, might interfere with the FcR dependent immunoregulation.
Human IgG Fc fragment is known to stimulate both human and mice B cells to differentiation into antibody secreting cells, although the mechanism of the activation is not completely clear (Berman ef al., 1978, Morgan and Weigle, 1981). In the present experiments we have tested the direct and indirect effects of Fc fragment and that of synthetic peptides comprising surface exposed sequences within the CH2 and CH3 domain of IgG on different steps of lymphocyte activation. A CH2 and a CH3 domain peptide as well as the whole Fc fragment were shown to enhance the IgM synthesis of PWM or PMA + CaI activated lymphocytes. The whole IgG molecule had no effect, suggesting that the responsible groups become available only after the cleavage of the molecule. Similar changes might occur after immunocomplex formation inducing a structural change in the Fc part. These observations suggest that after the binding of immune comlexes to FcR on different cells, low mol. wt peptides might be released by enzymatic cleavage, possibly influencing the function of immune competent cells. Fc fragment and Fc peptides were without any effect when added to the cultures following stimulation by PWM, suggesting that they exert their effect at the early phases of B cell stimulation. For optimal response to PWM, macrophages and T cells are required. We have, therefore, tested the Fc fragment and peptides using PMA + CaI as activation stimuli as well, because this response is not dependent on the presence of accessory cells. PMA activates protein kinase C directly on the surface of B cells and triggers the cells to enter to the Gl phase. In the presence of CaI the stimulation proceeds and the cells proliferate and differentiate into plasma cells. On the other hand, it has been demonstrated that PMA will act on a subpopulation of B cells which has already received an early activation signal (Dougas et al., 1986). Fc fragment and Fc peptides enhanced the moderate immunoglobulin production of B lymphocytes stimulated with PMA + Cal, suggesting that in interacting with B cells these reagents might be providing an early signal of activation, thereby rendering the cells more susceptible to the PMA stimulus. The role of monocytes cannot be completely ruled out in this process, however, since the separated B cell fraction was contaminated with a small percentage of monocytes (usually less than 3%). These results suggested that we should test the effect of Fc fragment and peptides on the early phases of B cell stimulation. When a separated resting subset of B lymphocytes was tested after short incubation with the Fc fragment or CH2 domain peptides, an increase in the cell volume and in the expression of HLA-DR antigen was observed, while peptides corresponding to sequences within the CH3 domain failed to induce this phenomenon. On the other hand Y75, the CH3 domain peptide, as well as the CH2 domain peptides and the whole Fc fragment,
Immunomodulatory
effect of synthetic peptides
induced LIF production of resting B cells. These early signs of activation are known to appear after a few hours of stimuiation and are characteristic of the early Gl phase of cell cycle (Szigeti, 1985; Kehrl et al., 1985; Gordon and Guy, 1987). These results suggest that the Fc fragment and Fc peptides are able to interact directly with resting B cells and induce them to leave Go. Since a significant proliferation was never observed in these cases, even in the presence of B cell stimulatory factors, probably additional signals are required for the cells to enter into the further phases. The direct interaction of Fc fragment and Fc peptides with the T subset did not induce the activation of these cells. since neither LIF and IL-2 production nor IL-2 receptor expression was observed. In cion activated B lymphocytes separated from the resting subset by Percoll gradient centrifugation also failed to show any response to the Fc fragment or peptides, suggesting that these products can directly interact with and stimulate specifically the resting B cell subpopulation. In order to test the possibility that Fc fragment and Fc peptides might induce the release of factors influencing B cell stimulation, supernatants of the peptide treated cells were tested for the presence of IL-I and IL-2. Fc fragment and immunocomplexes are known to induce IL-l production by macrophages (Oppenheim, 1986). In the supernatant of peptide treated PBMC a low amount of It-l, but no IL-2 was detected. The supernatants of adherent cells (monocytes) incubated in the presence of Fc fragment or Fc peptides overnight contained a significant amount of IL-l, while that of the monocyte depleted fraction failed to induce thymocyte proliferation. These results suggest that the peptides as well as the Fc fragment induce IL-1 production by monocytes. The lack of IL-2, and the low level of IL-l in PBMC supernatants can be explained probably by their consumption by the T cells. IL-l was shown to possess a B cell differentiation inducing activity as cofactor with other stimuli (Oppenheim, 1986, Gordon and Guy, 1987), thus the induction of IL-1 by the Fc peptides might be one of the explanations for their IgM synthesis enhancing effect when the B cells were triggered with additional stimuli (PWM or PMA + Cal). In the case of PWM stimulation in the presence of Fc fragment or peptides. the role of other factors released from T cells in response to IL-1 cannot be ruled out. Comparing the two CH2 domain peptides Y48 and Y91. the latter did not contain the 289Thr-292Arg residues representing the sequence of tuftsin, a known immunoregulatory peptide (Najjar, 1985, Florentin et al., 1986). Since both Y48 and Y91 were equally active in all tests, the residues corresponding to tuftsin cannot be responsible for the demonstrated effects. The CH3 domain peptide Y75 induced an enhanced IgM synthesis, LIF and IL-1 production, but did not enhance HLA-DR expression of resting B
1187
cells, suggesting that it acts mainly by the mediation of factors released from monocytes and B cells. The possible influence of this latter peptide on the in t&o response of mice to sheep erythrocytes or to the T cell dependent contact sensitizing hapten oxazolone, was tested earlier in our laboratory. Peptide Y75 significantly enhanced the immune response to SRBC and to both the primary and the secondary response against oxazolone (Gergely, 1985; Kulics et al., submitted for publication) suggesting that this peptide is able to exert its stimulating effect in z&o as well.
Taken together our results suggest that the peptides corresponding to surface exposed sequences within the CH2 or CH3 domain of IgG are able to enhance antibody production by activated cells. These peptides can exert their effect, on the one hand, directly by activating resting B lymphocytes and rendering these cells more susceptible to other stimuli and, on the other hand, they can enhance the immune response by triggering the release of factor(s) (e.g. IL-I) which act as cofactor(s) during B cell activation. Since similar peptides might be released % z&o as well, after the enzymatic cleavage of immune complexes, they might play an important regulatory role during the immune response. Ackno~~edg~me~l~-The authors are grateful to Baron Maximilian de Clara for generous financial support of certain aspects of this work.
REFERENCES
Berman M. A., Spiegelberg H. L. and Weigle W. 0. (1978) Lymphocyte stimulation with Fc fragments. I. Class, subclass and domain of active fragments. J. Immun. 122, 89-93. Dougas B., Vazquez A., Klein B., Delfraissy J-F., Rammou J-P., Gerard J-P. and Galanaud P. (1986) Early events in human B cell activation: metabolic pathways vary according to the first signal used. Eur. J. Immzm. 16, 1609-1614. Erdei A., Benczur M.. Fabry Zs., Dierich M. P. and Gergely J. (1984) C3 cleaved by membrane proteases binds to C3b acceptors expressed on Con-A stimulated human lymphocytes and enhances ADCC. Stand. J. Immun. 20, 125-133. Florentin I., Chung V., Martinez J., Maral J., Le Garret Y. and Mathe G. (1986) In rizw immunopharmacological properties of Tuftsin (Thr-Lys-ProArg) and some analogues. Mefh. Find. Expi. Clin. Pharmac. 8(2i, 73-80. Fruehauf J. P., Bonnard 6. D. and Herberman R. B. (1982) The effect of lentinan on production of interleukin-1 by human monocytes. lmmunopharmacology 5, G--74. Gergely J., Sarmay G.. Kulics J., Klein E. and Stanworth D. R. (1986) Peptides as immunomodulators. In Proceedings of lhc Xlll Congress off‘rho Europcwn Academy of Aliergolog~ and Clinicuf irnrnunolt~~~ (Edited by Csaba, B., Leovey A. and Szemere I’.), pp. 215-239. Alfoldi Nyomda. Debrecen. Hungary. Gillis S. and Smith K. A. f 1977) Long term culture of tumor specific cytotoxic T cells. Nature, Land. 268, 154-f 56. Gordon J. and Guy G. R. (1987) The molecules controlling B lymphocytes. Immun. Today 8, 339-344. Hobbs M. V., Morgan E. L. and Weigle W. 0. (1985) Synthetic Fc peptide-mediated regulation of the immune
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response, II. Analysis of secreted immunoglobulin classes, accessory cell contribution, and lymphocyte proliferation in p23-stimulated spleen cell cultures. J, Immun. 134, 2847-2852. Hobbs M. W., Morgan E. L., Houghten R. A.. Thoman M. L. and Weigfe W. 0. (1987) Identification ofa lymphocyte activating pentapeptide sequence in the Fc region of human IgGl. J. Immun. 138, 2581 -2586. Kehrl J. H., Muraguchi A. and Fauci A. S. (1985) The modulation of membrane Ia on human B lymphocytes. Ceil Immun. 92, 391.-403. Kolsch E., Oberbarnscheidt J., Bruner K. and Heuer J. (1980) The Fc receptor: its role in the transmission of differentiation signals. immun. Rev. 49, 61 78. Kulics J., Hilbert A., Stanworth D. R. and Gergely J. (1988) Synthetic human gamma chain peptide enhances in rite antibody responses in mice (submitted for publication). Martinez J. and Winter&z F. (1985) New synthetic and natural Tuftsin-related compounds and evaluation of their phagocytosis-stimulating activity. Ann. 1Y.Y. Actrri. Sci. 419. 23-34. Morgan E. L., Hugli T. E. and Weigle W. 0. (1982) Isolation and identification of a biologically active peptide derived from the CH3 domain of human IgGl. Proc. natn. Awd. Sri., U.S.A. 79, 5388-5390. Morgan E. L., Shields J. E., Campbell C. S.. Barton R. L.. Koppel G. A. and Weigle W. 0. (1983) Synthetic Fc peptide mediated regulation of the immune response. I. Characterization of the immunomodulating properties of a svnthetic 23-amino acid peptide derived from the sequince of the CH3 domain’of’human IgCl. J. esp. Med. 157, 9477952. Morgan E. L. and Weigle W. 0. (19X1} Polyclona! activation of human B lymphocytes by Fc fragments. 1. Characterization of the cellular requirement for Fc fragment-mediated polyclonal antibody secretion by human peripheral blood B lymphocytes. J. esp. Med. 154, 778-782. Najjar V. A. (1985) Tuftsin (ThrLys-Pro Arg): a natural activator of phagocytic cells with antibacterial and antineoplatic activity. In Biological Respwue Mod$ers. New
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Approaches to Diseuse Intervention (Edited by P. F. Torrence), p. 141. Academic Press, New York. Oppenheim, J. J., Kovacs E. J., Matsushima K. and Durum S. K. (1986) There is more than one interleukin 1. lrnmzm. Today -I, 45-56. Ramos 0. F., &may G., Klein E., Ycfcnof E. and Gergely J. (1985) Complement dependent cellular cytotoxicity: lymphoblastoid cell lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte mediated lysis in the presence of fresh human serum. Proc. mtn. Acud. Sci.. U.S.A. 87, 547@5474. Sarmay G.. Benczur M.. Petranyi Gy.. Klein E., Kahn M., Stanworth D. R. and Gergely J. (1984) Ligand inhibition studies on the role of Fc receptors (FcR) in antibody dependent cellular citotoxicity (ADCC). M&c. Imniun. 21, 43351. Sarmay G., Stanworth D. R., Szigeti R., Klein E., Reguly K. C ~1988) and Gergelv _ . J. The effect of synthetic peptides corresponding to Fc sequences in human IgG I on various steps in the B cell activation pathway. EEU. J. I~mzutt. 18, 2899294. Sinclair N. R. StC. and Panoskaltsis A. (1987) Immunoregulation by Fc signals. Immun. Today 8, 7679. Stanworth D. R. (1982) Application of synthetic peptides representative of immunoglobulin sequences to the delineation of receptor binding and signaling process. iMofcc. rrnrn~~i. 19, 1245--l 254. Szigeti R. (1985) Application of migration inhibitory techniques in tumor immunology. A&. C‘uncer Rex. 43, 241-270. llher F. and Dickler H. B. (1986) Cooperativity between B lymphocyte membrane molecules: independent ligand occupancy and cross-linking of antigen receptors and Fc receptors down-regulates B lymphocyte function. J. immun. 137, 31243129. Veretennikova N. I., Chipens G. I., Nikiforovich G. V. and Betinsh Y. A. R. (1981) Rigin, another phagocytosis stimulating tetrapapetide isolated from human IgG. Confirmations of a hypothesis. Int. J. Peptide Protein Res. 17, 430436.
0161.5890!88$3.00f0.00 Pergamon Press plc
IMMUNOMOD~LATORY EFFECT OF SYNTHETIC PEPTIDES CORRESFONDIN~ TO SEQUENCES WITHIN THE CH2 AND CH3 DOMAIN OF HUMAN IgGl GABRIELLA SARMAY,* KRISZTINA REGULY,* ROBERT SZICIETI,~ EVA KLEIN,? DENIS R. STANWORTH: and JANOS GERGELY* *Department of Immunology, L. E6tv8s University, G6d. Hungary: TDepartment of Tumor Karolinska
Institutet,
Stockholm,
Sweden; and $Department U.K.
of Immunology.
University
Biology, of Birmingham.
Fc region of IgG is known to be the source of small peptides possessing immunomodulatory function. Results are summarized showing the effect of synthetic peptides composed of surface exposed residues of Cy2 or Cy3 domains on different steps of human B lymphocyte activation
Abstract-The
cycle. Both the CH2 (289Thr-3OIArg) and CH3 (4~7Tyr~~6Arg) peptides as well as the whole Fe fragment enhanced the IgM synthesis of PWM or PMA + CaI activated lymphocytes. This effect was exerted at the early phase of B cell activation. The incubation of separated resting B cells with Fc fragments or CH2 peptides resulted in increase of cell volume and in expression of HLA-DR antigen. On the other hand, LIF production was induced both by CH2 and CH3 pcptides. It was also shown that Fe peptides induce IL-1 release from monocytes. The results suggest that the CHZ and CH3 domain peptides exert their effect partly directly, by activating resting B cells, rendering the cells more susceptible to other stimuli: and moreover. by enhancing the humoral response by triggering the release of IL-I.
INTRODUCTION
Immunoglobulins secreted by activated B cells have a multipotential role in regulating the immune response. Antigen-~omplexed IgG molecules are able to bind to specific receptors (FcR)* on the surface of different cell types, thereby in~uencing functions such as phagocytosis of macrophages, antibody dependent cellular cytotoxicity (ADCC) of K cells and regulation of Ig synthesis by B cells. Both the enhancement and the inhibition of antibody production induced by IgG molecules or its fragments has been described (Sinclair and Panoskaltsis, 1987). Crossbinding of sIg and FcR on the surface of the same B cell results in the inhibition of Ig synthesis (Kolsch et al., 1980, Uher and Dickler, 1986). On the other hand, Fc fragment of IgG molecule and a peptide (~23) released from the Cy3 domain by surface enzymes of macrophages have been shown to stimulate the antibody productjon of mice spleen ceils (Morgan et al., 1983, Hobbs er al., 1985). Recent findings suggest that various small mol. wt peptides possessing immunomodulatory function _____~_. --__-_. *List of abbreviations:
ADCC, antibody dependent cellular cytotoxicity; Cal, Ca ionophore A23187; FcR, receptors interacting with the Fc portion of IgG; FITC, Auorescein-isothiocyanate; IL-I, interleukin-1; IL-2. interleukin-2; LIF, leukocyte migration inhibitory factor; MHC, main histocompatibiiity complex; PBMC, peripheral blood mononuclear cells; PHA, phytohaemaglutin~n~ PMA, phorbol-12 myristate-13 acetate; PWM, pokeweed mitogen; sIg. surface immunoglobulins. §Correspondence to be addressed to: Gabriella Sarmay, Department of Immunology, L. EEitviis University, Javorka S.U. 14, Gild, 2131 Hungary.
might be released from the Ig molecules in citv. Tuftsin (289Thr--292Arg), one of the best characterized peptides amongst these influences mainly the function of macrophages (Najjar, 1985; Florentin et al., 1986). A Cy 3 domain peptide (345Glu-349Arg) called rigin has also been shown to have a tuftsin-like effect (Veretennikova et al., 1981). More recently, a ~ntapeptide (351 Leu-355Arg) was found to be capable of demonstrating the immunostimulato~ effect of the peptide p23 (Hobbs et al., 1987). A decapeptide representative of a sequence within the CH4 domain of the human IgE molecule has been shown to induce histamine release from mast cells in a manner closely resembling the IgE dependent triggering process (Stanworth, 1982). Since some of these peptides has been shown to be released in a&o as well, one may predict that they are localized on the surface of Ig molecule, where they would be available for enzymatic cleavage. On the basis of their hydrophilicity and accessibility, groupings of certain amino acid residues within the IgG Fc region can be predicted to constitute antigenic epitopes on the surface of the Cy2 and Cy 3 domains (Martinez and Winternitz, 1983). These sites are available for interactions with other cell surface receptors as well as being vulnerable to enzymatic cleavage. Some of these surface exposed hydrophilic patches, like those composed of residues 285-293 in the CH2 or 33 l--346 in the CH3 domains of IgG molecules incorporate sequences corresponding to peptides with known immunoregulatory properties. Taking into consideration these facts, we have planned and synthesized peptides comprising further
1183
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GABRIELLA S~RMAY
et al.
Table I. Location of synthetic peptides on IgGl Fc Peptides
Sequences
CH2 domain: Y48 Y91
(289)Thr-LysPro-Arg-Gl~Gl~Gl~Tyr-AsSer-Thr-Tyr~Arg(3~~) (293)GI~GlbGI*Tyr-Asp%-Thr-TyrArg(3Ol)
CH3 domain: Y75
(407)Tyr-SermLys-LewThr-V&AspLys-Ser-Arg(4l6)
Bold characters stand for the predicted antigenic epitopes of IgGl (Martinez and Winter&z, 1983)
surface exposed sequences of the IgG molecule and tested their effects on different steps of the human lympocyte activation cycle. RESULTS
Synthetic peptides composed of surjtice exposed residues of Cy 2 or Cy 3 domains enhance IgM synthesis by stimulated human B lymphocytes The synthetic peptides studied in the following experiments are listed in Table 1. Peptide Y48 includes the sequence corresponding to tuftsin, while peptide Y91 represents the sequence downstream from the tuftsin. Peptide Y75 contains hydrophilic residues of the CH3 domain and is different from the one studied earlier by others (Hobbs et al., 1987). Peptides Y48, Y91 and an analogue of Y75 have been found previously to inhibit the FcR mediated ADCC (S&may et al., 1984). Some of the results presented here have been published in detail elsewhere (Sdrmay et al., 1988). The influence of the peptides and that of the Fc fragment or the whole IgG molecule on B cell stimulation was studied first using the in vitro cultures of peripheral blood mononuclear cells activated with suboptimal amount of pokeweed mitogen (PWM). The proliferation of these cells was not affected by Fc fragment or Fc peptides, whereas they significantly enhanced IgM synthesis of activated B cells as detected in the supernatants of the cultures by ELISA (Table 2). In contrast, the whole IgG molecule had no effect. The peptides or Fc fragment alone, in the absence of PWM, failed to induce either proliferation or antibody synthesis (data not shown). Both the mitogen and the Fc peptides needed to be present from the initiation of the culture in order to induce an optimal enhancement of IgM secretion, whereas, adding them at the third day had no significant effect.
The response to PWM is macrophage and T cell dependent. Therefore, in further experiments, separated B cells were stimulated directly by phorbol-12myristate- 13-acetate (PMA) plus Ca ionophore A23187 (Car) in the presence and in the absence of peptides, and IgM secretion into the supernatant of the cultures was compared. Fc fragment and Fc peptides induced an enhanced IgM synthesis compared to the samples stimulated with PMA + CaI alone. Fc fragment induced some IgM synthesis in the absence of additional stimuli as well (Fig. 1). Fc peptides induce interleukin-1 (IL-l) monocytes
Fc fragment was previously shown to induce IL-l production by macrophages (Oppenheim et al., 1986). Adherent cells from peripheral blood monocular cells were cultured in the presence of Fc frag100
r
i FC
[il lbl Y48
content of supernatant was measured by ELISA assay. The first columns represent the samples cultured without PMA and CaI, the second columns demonstrate the IgM level in stimulated cultures.
IgM content of the supernatants (ng/ml) YlS Y48 Y91
FC
1st day 3rd day
985 f 305 nt
798 f 30 327 + 30
903 i 30 432 + 50
726% 15 380 F 40
5
1st day 3rd day
610 + 101 nt
482 f 100 272 f IO
449 + 100 326 F 35
390 f 40 2X4 k 80
0.5
1st day 3rd day
465 k 63 “t
318 + 60 231 + 80
340 f 20 25X k 60
324 + 40 205 f 70
Control:
Y75
Fig. 1. Fc peptides and Fc fragment enhance IgM synthesis induced by PMA plus Ca ionophore A23187. Separated B cells were stimulated by 1ng/ml PMA and 0.5pg/ml Ca ionophore in the presence and in the absence of Fc fragment or Fc peptides (10 ,~g/2 x 10S cells) for 8 days. The IgM
Table 2. Fc fragment and synthetic peptides corresponding to sequences withm the CH2 or CH3 domain of IgGl enhance PWM driven IgM synthesis Peptide doses (pglml) 50
release from
244 f 90 ng/ml
Fc fragment or Fc peptides were added either at the initiation OTcultures together with PWM (1st day), or after 3 days stimulation with PWM. The IgM content was measured by ELISA assay on the 9th day. Mean k S.E. of 6 experiments.
Immunomodulatory
effect of synthetic
. . I
. .
4 . :
. . Ic
:
.
.
-+
L
I
I
I
I
I
Fab
Fc
Y48
Y91
Y75
Fig. 2. Fc peptides as well as Fc fragment induce IL-I production by monocytes. Adherent cells were purified from PBMC in 24 well Costar plates by incubation of 5 million cells in 1ml complete medium at 37°C for 2 hr followed by the vigorous washing of the non-adherent population. Fifty pg Fc fragment or peptides were added to the cultures of 1 ml medium, and the samples were incubated at 37°C for 18 hr. The supernatants were harvested and tested in the standard thymocyte-costimulatory assay. The results are expressed as stimulation indices: [‘HI thymidine incorporation in test supernatants divided by that of the control cultures incubated in medium alone.
ment and Fc peptides for 18 hr, and the supernatants of cultures were tested in the standard thymocyte co-stimulatory assay (Fruehauf et al., 1982). Both the Fc fragment and the Fc peptides induced IL-1 synthesis by monocytes. The mean stimulation indices were 2, 1.6 and 2.1 for Fc fragment, Y48 and Y75 respectively. Fab fragment did not induce IL-l production. The amount of IL-l detected in the supernatants of Fc fragment or peptide treated monocytes corresponded to 400U/ml IL-l compared to thymocyte proliferation induced by recombinant IL-l standard (Fig. 2). In the supernatant of PBMC cultured in the presence of peptides only a moderate, insignificant, amount of IL-1 was detected, probably because of the consumption of the synthesized IL-I by the cultured cells. Supernatants of the monocyte depleted cultures had no effect on thymocyte proliferation (data not shown). The Fc peptide induced supernatants were also tested for the presence of interleukin-2 (IL-2). The 48 hr supernatants of PBMC cultured with Fc fragment for Fc peptides did not contain a detectable amount of IL-2 as assayed in the standard IL-2 dependent CTLL proliferation assay (Gillis and Smith, 1977) (data not shown). IL-2 receptor expression has also not been detected on the peptide treated T cells. The effect oj’Fc,fragments and Fcpeptides on the early signs of B cell stimulation B lymphocytes have been shown to undergo some rapid changes after being exposed to different stimuli.
1185
peptides
For instance, they might produce leukocyte migration inhibitory factors (LIF), express a higher amount of MHC class II antigen on their surface and increase in volume (Szigeti, 1985; Kehrl et al., 1985). To test the possibility that Fc fragment and peptides might have a direct effect on B lymphocytes, the resting B subset was separated by Percoll gradient centrifugation, and cultured in the presence of these reagents for 18 hr. The supernatants of these cells were tested for the presence of LIF in a granulocyte migration inhibitory assay. while the resting B lymphocytes from the same cultures were assayed for the of HLA-DR antigen by indirect expression immunofluorescence. Both Fc fragment and Fc peptides induced dose dependently LIF production by resting B, but not by T lymphocytes (Fig. 3) nor by already activated B cells (data not shown). The amount of LIF induced by the peptides or Fc fragment was comparable to that measured in the anti-IgM stimulated B cell cultures. T lymphocytes produced LIF when stimulated with PHA (Fig. 3). The expression of HLA-DR antigen on resting B cells was significantly enhanced as a result of culturing them overnight with Fc fragment or Y48 and Y91 peptides compared to control samples, whereas peptide Y75, the CH3 domain peptide had no effect. The volume of the positive cells also increased in the former cases. Figure 4 demonstrates a representative experiment obtained with Fc fragment and peptide Y48. Taken together these findings indicate that Fc fragment and synthetic peptides corresponding to surface exposed sequences within the CH2 or CH3 domain are able to induce the appearance of certain early signs of activation on resting B lymphocytes, although peptides comprising sequences from the CH2 and CH3 domain differed in respect to their effect on HLA-DR expression.
c
c
Fob
FC
Y49
Y91
Y75
PHA Antim
Fig. 3. LIF production by separated resting B (N) and T (0) lymphocytes cultured with Fc fragment (30 pg/ml), Fc peptides (100 pg/ml), anti-IgM (30 pg/ml) and PHA (2 pg/ml) respectively, overnight. Supernatants of the samples were tested by indirect leukocyte migration inhibitory assay. Control samples were treated with medium only. Mean + SE. of 8 experiments.
GABRIELLA SARMAY et al.
1186
Channel
number
Fig. 4. Increase of the cell volume and HLA-DR expression on resting B lymphocytes cultured with Fc fragment or the CH2 domain peptide Y48. Control samples were cultured in medium only. The cells were treated with monoclonal antibody specific for HLA-DR. then with FITC-conjugated anti-mouse Ig. The samples were analysed by FACS.
DISCUSSION
Low mol. wt peptides from various protein molecules might be released by surface enzymes of different cell types. Such enzyme activity has been detected on macrophages, activated T cells and lymphoblastoid B cell lines (Morgan et al., 1982; Erdei et al., 1984; Ramos et al., 1985). The cleavage of the IgG molecule following its binding to FcR on macrophages was demonstrated earlier (Morgan er al., 1983). Peptides corresponding to surface exposed sequences of immunoglobulins has been shown to possess an immunomodulatory effect (Martinez and Winternitz, 1985). Synthetic peptides comprising sequences within the CH2 and CH3 domain of IgGl were previously found to inhibit ADCC suggesting, although only indirectly, that they might bind to the receptors specific for the IgG Fc (Sarmay et ul.. 1984). On the other hand, the possibility that the Fc peptides act through binding to other structures on the cell surface cannot be excluded. FcR play an important role in the regulation of antibody production. Depending on the interactions of ligands with FcR or antigen receptors on the surface of B lymphocytes, the antibody synthesis can be up- or down-regulated (Sinclair and Panoskaltsis, 1987). Peptides originating from the Fc part of IgG molecules and possibly released in aivo as well, might interfere with the FcR dependent immunoregulation.
Human IgG Fc fragment is known to stimulate both human and mice B cells to differentiation into antibody secreting cells, although the mechanism of the activation is not completely clear (Berman ef al., 1978, Morgan and Weigle, 1981). In the present experiments we have tested the direct and indirect effects of Fc fragment and that of synthetic peptides comprising surface exposed sequences within the CH2 and CH3 domain of IgG on different steps of lymphocyte activation. A CH2 and a CH3 domain peptide as well as the whole Fc fragment were shown to enhance the IgM synthesis of PWM or PMA + CaI activated lymphocytes. The whole IgG molecule had no effect, suggesting that the responsible groups become available only after the cleavage of the molecule. Similar changes might occur after immunocomplex formation inducing a structural change in the Fc part. These observations suggest that after the binding of immune comlexes to FcR on different cells, low mol. wt peptides might be released by enzymatic cleavage, possibly influencing the function of immune competent cells. Fc fragment and Fc peptides were without any effect when added to the cultures following stimulation by PWM, suggesting that they exert their effect at the early phases of B cell stimulation. For optimal response to PWM, macrophages and T cells are required. We have, therefore, tested the Fc fragment and peptides using PMA + CaI as activation stimuli as well, because this response is not dependent on the presence of accessory cells. PMA activates protein kinase C directly on the surface of B cells and triggers the cells to enter to the Gl phase. In the presence of CaI the stimulation proceeds and the cells proliferate and differentiate into plasma cells. On the other hand, it has been demonstrated that PMA will act on a subpopulation of B cells which has already received an early activation signal (Dougas et al., 1986). Fc fragment and Fc peptides enhanced the moderate immunoglobulin production of B lymphocytes stimulated with PMA + Cal, suggesting that in interacting with B cells these reagents might be providing an early signal of activation, thereby rendering the cells more susceptible to the PMA stimulus. The role of monocytes cannot be completely ruled out in this process, however, since the separated B cell fraction was contaminated with a small percentage of monocytes (usually less than 3%). These results suggested that we should test the effect of Fc fragment and peptides on the early phases of B cell stimulation. When a separated resting subset of B lymphocytes was tested after short incubation with the Fc fragment or CH2 domain peptides, an increase in the cell volume and in the expression of HLA-DR antigen was observed, while peptides corresponding to sequences within the CH3 domain failed to induce this phenomenon. On the other hand Y75, the CH3 domain peptide, as well as the CH2 domain peptides and the whole Fc fragment,
Immunomodulatory
effect of synthetic peptides
induced LIF production of resting B cells. These early signs of activation are known to appear after a few hours of stimuiation and are characteristic of the early Gl phase of cell cycle (Szigeti, 1985; Kehrl et al., 1985; Gordon and Guy, 1987). These results suggest that the Fc fragment and Fc peptides are able to interact directly with resting B cells and induce them to leave Go. Since a significant proliferation was never observed in these cases, even in the presence of B cell stimulatory factors, probably additional signals are required for the cells to enter into the further phases. The direct interaction of Fc fragment and Fc peptides with the T subset did not induce the activation of these cells. since neither LIF and IL-2 production nor IL-2 receptor expression was observed. In cion activated B lymphocytes separated from the resting subset by Percoll gradient centrifugation also failed to show any response to the Fc fragment or peptides, suggesting that these products can directly interact with and stimulate specifically the resting B cell subpopulation. In order to test the possibility that Fc fragment and Fc peptides might induce the release of factors influencing B cell stimulation, supernatants of the peptide treated cells were tested for the presence of IL-I and IL-2. Fc fragment and immunocomplexes are known to induce IL-l production by macrophages (Oppenheim, 1986). In the supernatant of peptide treated PBMC a low amount of It-l, but no IL-2 was detected. The supernatants of adherent cells (monocytes) incubated in the presence of Fc fragment or Fc peptides overnight contained a significant amount of IL-l, while that of the monocyte depleted fraction failed to induce thymocyte proliferation. These results suggest that the peptides as well as the Fc fragment induce IL-1 production by monocytes. The lack of IL-2, and the low level of IL-l in PBMC supernatants can be explained probably by their consumption by the T cells. IL-l was shown to possess a B cell differentiation inducing activity as cofactor with other stimuli (Oppenheim, 1986, Gordon and Guy, 1987), thus the induction of IL-1 by the Fc peptides might be one of the explanations for their IgM synthesis enhancing effect when the B cells were triggered with additional stimuli (PWM or PMA + Cal). In the case of PWM stimulation in the presence of Fc fragment or peptides. the role of other factors released from T cells in response to IL-1 cannot be ruled out. Comparing the two CH2 domain peptides Y48 and Y91. the latter did not contain the 289Thr-292Arg residues representing the sequence of tuftsin, a known immunoregulatory peptide (Najjar, 1985, Florentin et al., 1986). Since both Y48 and Y91 were equally active in all tests, the residues corresponding to tuftsin cannot be responsible for the demonstrated effects. The CH3 domain peptide Y75 induced an enhanced IgM synthesis, LIF and IL-1 production, but did not enhance HLA-DR expression of resting B
1187
cells, suggesting that it acts mainly by the mediation of factors released from monocytes and B cells. The possible influence of this latter peptide on the in t&o response of mice to sheep erythrocytes or to the T cell dependent contact sensitizing hapten oxazolone, was tested earlier in our laboratory. Peptide Y75 significantly enhanced the immune response to SRBC and to both the primary and the secondary response against oxazolone (Gergely, 1985; Kulics et al., submitted for publication) suggesting that this peptide is able to exert its stimulating effect in z&o as well.
Taken together our results suggest that the peptides corresponding to surface exposed sequences within the CH2 or CH3 domain of IgG are able to enhance antibody production by activated cells. These peptides can exert their effect, on the one hand, directly by activating resting B lymphocytes and rendering these cells more susceptible to other stimuli and, on the other hand, they can enhance the immune response by triggering the release of factor(s) (e.g. IL-I) which act as cofactor(s) during B cell activation. Since similar peptides might be released % z&o as well, after the enzymatic cleavage of immune complexes, they might play an important regulatory role during the immune response. Ackno~~edg~me~l~-The authors are grateful to Baron Maximilian de Clara for generous financial support of certain aspects of this work.
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