Bioactive complement fragments in immunoregulation

Immunology Letters, 9 (1985) 207-213

Elsevier Imlet 617

BIOACTIVE C O M P L E M E N T F R A G M E N T S IN I M M U N O R E G U L A T I O N * E. L. MORGAN, M. L. T H O M A N , P. D. H O E P R I C H and T. E. H U G L I Department of Immunology. Scripps Clinic and Research Foundation, La Jolla, CA 92037, U.S.A.

(Received and accepted 18 February 1985)

1. Summary

2. Introduction

Several fragments derived from complement components have been identified as potent effector substances in in vitro assays that measure cell proliferation and antibody synthesis. The anaphylatoxin C3a suppresses the immune response but fails to influence T- or B-cell proliferation. The factor C5a augments both antibody production and antigen-induced, but not mitogen-induced, T-cell proliferation. C3a-mediated suppression occurs through the activation of a suppressor T-cell cascade with macrophage collaboration. C5a-mediated enhancement, depending upon the in vitro system studied, acts at the level of the helper T cell and/or macrophage. A fragment generated from treating iC3b with kallikrein (C3d-K) has aided in defining a structural region of the C3b molecule that can influence the level of circulating leukocytes. The factor C3d-K is also capable of suppressing both specific and non-specific T-cell proliferative responses and mitogen-induced B cell growth. The mechanism of C3d-K action is defined as a direct effect on "activated" T cells, even though IL-2 synthesis of treated cells is diminished. Fhe effect of C3d-K is long lasting, non-reversible md requires only a short exposure to the target cell.

Various aspects of the regulation of the immune response by complement have been extensively investigated [1]. The observation that leukocytes from a number of species possess receptors for a variety of complement fragments led to the concept of complement-mediated immunoregulation recently reviewed by Ross and Newman [2]. The nature of complement-mediated immunoregulation remains controversial as does the influence of complement on immune responses in vivo [3]. Confusion may be attributable in part to the lack of defined culture conditions, species differences and the use of ill-defined or poorly characterized reagents. To overcome some of these potential pitfalls, we examined the immunoregulatory properties of several highly purified complement-derived fragments under in vitro assay conditions, the anaphylatoxins C3a and C5a as well as the C3-derived fragment C3d-K were characterized. Regulatory properties of these fragments have been assessed in vitro in both proliferative and humoral immune response models.

3. Results 3.1. C3a-mediated suppression o f humoral immune responses

* To be presented at the "Immunobiolo#cs and Their Application" meeting,Budapest, Hungary,April 21-27, 1985. Key words: complementfragments-- immunoregulation

The C3a molecule has the capacity to suppress both specific and nonspecific humoral immune responses. Addition of human C3a to cultures of human PBL or murine splenic lymphocytes results in suppression of both the anti-SRBC response and the

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207

Table 1 Suppression of human and murine anti-SRBC responses by C3a Lymphocyte source

SRBC

C3a a

Human PBL + + Murine spleen

+ +

Direct anti-SRBC PFC (± SD)

+

25: I b 1335:2 95: I

+

535:2 c 16705:86 3405:1

a 25 #g C 3 a / m l ; 1 mM 2-mercaptomethyl-5-guanidinopentanoic acid present. b PFC/culture. c PFC/106 imput lymphocytes.

Fc fragment-mediated polyclonal antibody response (Table l) [4]. In contrast to these results, mitogenand antigen-induced T- and B-cell proliferative responses are not affected by C3a. Concentrations of C3a at levels 10-fold greater than those required to suppress fully the anti-SRBC response have no effect on the proliferative responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM). In addition, B-cell proliferation induced by Fc fragments of immunoglobulin or lipopolysaccharide (LPS) remains unaffected by C3a [4]. Immune suppression mediated by C3a occurs at an early phase in the antibody response. This conclusion came from our observation that when C3a is added on day 0, maximal suppression is achieved;

however, when added on day 1, only marginal suppression is observed [4]. In addition, the interaction of purified T cells with C3a for as little as 30 min results in a pronounced suppression of B cell proliferation. The carboxy-terminal arginine in C3a is essential for immunosuppressive activity [1-4]. This result is consistent with a dependence of the spasmogenic properties of C3a on the COOH-terminal arginine [5]. Taken together, our results indicate that the COOH-terminal portion is, as in other bioactivities of C3a, the active center for mediating immunosuppressive actions of the factor. Therefore, the serum carboxypeptidase (SCPN) serves the same role in controlling the immunosuppressive activities of C3a as it does in restricting spasmogenic action. Data derived using synthetic peptides based on the structure of human C3a map the active region of the molecule at the carboxy terminus (Table 2) [6]. T lymphocytes are concluded to be the target of C3a-mediated suppression of the immune response. Substitution of T cells by a soluble T-cell factor in the Fc fragment-induced polyclonal antibody response abrogates the suppressive response of C3a [4]. Additional evidence for T ceils being the target of C3a-mediated suppression is drawn from our observation that incubation of purified T cells with C3a renders the T cells incapable of helping in the Fc-induced polyclonal antibody response. Moreover, C3a fails to suppress in vitro antibody responses to Tcells independent antigens such as T N P - L P S or TNP-Ficoll.

Table 2 Relative potency of synthetic analogue peptides of C3a Peptide

Activitya (%)

Concentration b (M)

Native C3a (C3a 1-77) Native C3ades Arg (C3a 1 76) C3a 57 77 C3a 65-77 C3a 70-77 C3a 73-77 C3a 65 77-glycine

100 0 44 1.0 0.3 0.03 0

4.4× NA c 9.9x 4.3× 1.4× 1.4× NA

Iff 8 10 10 10 10

8 6 6 5

a Values represent the activity relative to native C3a. b Concentration required to suppress by 50% the Fc fragment-mediated polyclonal antibody production by human PBL. c Not active to 1.0X l0-5 M,

208

ff--£-q n

i

Fig. 1. The sites of action of complement fragments on the immune response are illustrated. Factor C3a acts at the level of T cells to suppress the T-dependent humoral immune response. C5a acts at the level of the T cell and/or macrophage to augment antigen-induced T cell proliferation and antibody section. Factor C3d-K acts to suppress T- and B-ceU proliferation and inhibits humoral immune responses.

Incubation of T cells with C3a ultimately results in the generation of Lyt2+ suppressor T cells [7]. The action of C3a-suppressor T cells appears to be nonspecific in nature because these ceils are capable of suppressing both anti-SRBC and Fc fragment-induced polyclonal antibody responses. The mechanism through which C3a activates suppressor inducer T cells is unknown (Fig. 1). The C3a either acts directly on a subpopulation of Lytl + T cells (suppressor inducer) or on macrophages which in turn release a mediator, perhaps a prostanoid, that activates the suppressor T cell cascade. As indicated in Fig. 1 the suppressive effect of C3a may be negated by interleukin-2 (IL-2) lending further support for the T cell being the target of C3a-mediated suppression. Generation of suppressor T cells requires the interaction of T cells, C3a and macrophages [7]. The exact role of macrophages is presently being explored. The quantity of human C3a needed to suppress human PBL responses is 100-fold lower than that needed to suppress immune responses using murine PBL. It is currently unknown whether species differences are expressions of receptor specificity or of

true variations in sensitivity between human and murine helper T cell populations. It is clear that the level of C3a required to suppress the antibody response falls well within the physiologic concentration range in man. Significant suppression of responses by human PBL is observed at C3a concentrations of 0.1-1 #g/ml, levels well below the potential 50-60 ~tg/ml of C3a that may be generated by maximal conversion of human C3. In addition to the immunosuppressive properties of C3a described here, C3a has recently been shown to reduce generation of leukocyte inhibitory factor (LIF) by human T cells cultured with mitogens or antigens. Moreover, a synthetic C3a octapeptide, C3a 70-77, was found to reduce LIF levels. Reduction of LIF production by C3a is not mediated through a reduction in T-cell proliferation. Neither C3a nor C3a 70-77 altered the [3H]TdR uptake by human T cells exposed to mitogen or antigen [8]. 3.2. C5a-mediated enhancement o f the immune response

Human C5a has the ability to potentiate both antigen-specific and mitogen-induced humoral immune responses in either human or murine cell systems (Table 3) [1,9]. Addition of either C5a or the des Arg form of the molecule to human PBL cultures results in enhancement of both the anti-SRBC response and the Fc fragment-mediated polyclonal antibody response. These results are in agreement with the report of Goodman et al. [10] who showed that the murine anti-SRBC response is enhanced by human C5a and C5ades Arg. The specific T-cell proliferative response to tetanus toxoid is also augmented by Table 3 C5a-mediated enhancement of the in vitro anti-SRBC response by human PBL Stimulator a

S R BC

C5a C5ades Arg

+ + +

-

Direct anti-S R BC PFC/culture b (± SD) 30+

9

150± 15 6755:52 620± 15

a 1.0 #g/ml. b I mM 2-mercaptomethyl-5-guanidinopentanoic acid was present in all cultures.

209

C5ades Arg" These results are in contrast to those obtained with mitogen-induced proliferative responses. Concentrations of C5ade~Arg capable of augmenting antigen-induced proliferation have no effect on the P H A - or PWM-induced proliferative responses. The C-terminal arginine is C5a is not essential for this molecule to exhibit immunopotentiating properties. Removal of the terminal arginine by (SCPN) exerts only a minor effect on the ability of the molecule to augment nonspecific or specific humoral immune responses. These results are consistent with the finding that when human C5a is converted to C5ades Arg the spasmogenic action is largely lost; however, C5ades Arg retains an ability to promote other responses such as neutrophil chemotaxis and degranulation. Both C5a and C5%eSArg appear to be equally effective as augmentors of specific and nonspecific humoral immune responses. In contrast, C5ades Arg was found to be 10- to 30-fold less active than C5a when assessed for neutrophil chemotactic activity as observed by [11] ourselves and by others. T lymphocytes are involved in C5a-mediated enhancement of the human immune response. Replacement of T cells by a soluble human T-cell factor, Fc-TRF, in the Fc fragment-induced polyclonal antibody response abrogates the potentiating activity of C5ades Arg" The influence of C5a on T cells could be mediated through T-cell-macrophage interactions and the reason that mitogen-induced proliferative responses are not enchanced by C5a may be that the macrophage requirements are different from those of antigen-induced responses. In addition, C5a is unable to augment in vitro antibody responses to either T N P - L P S or TNP-FicoI1.

3.3. Significance of anaphylatoxin-induced immuno-

regulation The physiologic significance of C3a- and C5a-mediated immunoregulation is a matter of speculation. Regulation of immune responses by complement components may form a nonspecific in vivo immunoregulatory network. Generation of C3a in circulation by either the classical or alternate pathway of complement may potentiate nonspecific suppressor circuits capable of reducing both ongoing humoral and T-cell-mediated responses (Fig. 2). Although C3a is relatively short-lived in circulation, the fact that suppression is mediated by a C3a-induced sup210

Complement Activation ,

?

Lyt 1+

Lyt I +

f.--~Lyt 1+

He~per )

ovleLrri2des_~ ~

C3a Effect

~

(

DecreasedAntibodyProduction Fig. 2. Proposed mechanism for C3a-induced immunosuppression. C3a either acts directly on or in collaboration with macrophages to elicit activation of a nonspecific Lyt2 + suppressor T-cell circuit. The suppressor T cells appear to act by down-regulating helper-cell activity. The end result of the C3a effect on T-dependent B-cell function is reduced antibody production. Further evidence for the T cell being the target of C3a-mediated suppression comes from the finding that IL-2 can effectively override the suppressive effect of C3a.

pressor cell suggests a potential action in vivo for this labile hormone. The presence of SCPN in serum acts as a control mechanism for C3a suppressive action. However, production of relatively high concentrations of C3a in a microenvironment of interacting T cells, B cells, and accessory cells could lead to significant activation of suppressor T cells before the C3a is inactivated by the carboxypeptidase. In contrast, generation of C5a appears to activate a potent nonspecific enhancement network involving macrophages which affects antibody responses to T-cell dependent antigens. The ability of C5a to augment the antibody response overrides the suppressive effects of C3a (Fig. 2). Although these are opposing actions there are instances where C3a is generated without evidence for significant C5a formation, such as complement activation by circulating immune complexes, and this would result in only the suppressive effect of the anaphylatoxin. Thus, there appears to be a complex interplay between complement peptides and various cellular components in regulating the cellular immune response. 3.4. C3b-derived immunomodulators Several proteolytic cleavage products of C3b have

ditionally, they showed that C3d-K inhibits IL-2mediated T-cell mitogenesis, possibly by blocking synthesis of the lymphokine. These results indicate that C3d-K suppression of cellular proliferation is not limited to lymphocytic T cells only. A second function associated with C3d-K is that of inducing leukocytosis, i.e. an increase in the number of leukocytes in peripheral blood. Meuth et al. [12] showed that an i.v. injection of I nmol C3dK / k g body weight caused an appreciable increase in the number of circulating leukocytes in the rabbit model. Leukocytosis activity has been previously associated with two other C3-derived fragments; namely C3e [14] and leukocyte mobilizing factor ( L M F ) [15]. These latter two factors are anionic and of similar size ( M r = 10,000). They are thought to arise during the final stages of iC3b catabolism. It is speculated that the C3d-K fragment contains part or all of these two smaller fragments. A C3-derived polypeptide generated when Factor I inactivates cell b o u n d C3b, called C3d,g (Fig. 3), is virtually identical to C3d-K in size and function except that C3d,g fails to induce leukocytosis. The NH2-terminal sequence of C3d,g overlaps with

been reported to influence humoral and cell-mediated immune responses. A n active fragment recently described was C3d-K, a 40,000-Da polypeptide generated by treatment of iC3b with h u m a n plasma kallikrein (see model of C3 in Fig. 3). This fragment is an effective inhibitor of T cell proliferation and induces leukocytosis when injected intravenously into rabbits. Meuth et al. [12] showed that the addition of C3d-K to P H A - i n d u c e d cultures of PBL suppresses lymphocyte proliferation. Similarly, the fragment suppresses antigen-induced T-lymphocyte proliferation. Addition of C3d-K to h u m a n spleen cell cultures containing tetanus toxoid resulted in a marked reduction of cell growth. In the latter study, 50% suppression was attained with a quantity of C3d-K equivalent to the conversion of only 0.2% of normal circulating levels of C3. The negative effect on mitogen- and antigen-induced proliferation shows that C3d-K can suppress both specific and non-specific T-cell responses. M o r e recently, T h o m a n et al. [13] have shown that C3d-K can suppress B-cell mitogenesis and spontaneous growth of certain t u m o r cell lines. Ad-

C3a

C3b I I

C3 Convertase

S

S

K I E,T ~ d : ~ : ig ~ ~ : i ;

]~-N°napeptide7 S~S

"5,000 "C3g C3d

COOHa

Chain

2,000

C3d,g

I C3dK HOOC" ~ . ~ ~ i ~ : ' 7 " 5 : 5 ~ . . ' . : i ~

T ]- NHz/~ Chain

Fig. 3. A schematic model of human C3 is provided to indicate the location of C3d-K fragment in the molecular structure. C3 is initially cleaved into C3a and C3b by the serum convertases. Cleavage sites for enzymes other than the convertase are indicated on the a-chain. These enzymes are C3b inactivator (1), elastase (E), trypsin (T) and kallikrein (K). In serum, C3b is processed by C3b inactivator to C3c and C3d,g, with C3d,g being cleaved subsequently to C3d and C3g. Purified C3b can be converted to iC3b by C3b inactlvator and kallikrein is then used to generate the 40,000-Da fragment C3d-K. The solid region at the NH2-terminal end of C3dK represents the nonapeptide sequence (TLDPERLGR) which is the apparent sequence difference between C3d,g and C3d-K. Disulfide linkages, polysaccharide sites (~) and molecular weight assignments of the various a-chain fragments were based in part on the C3 gene structure reported by M. de Bruijn and G. Fey (PNAS, in press (1985)). 211

C3d-K beginning at residue 10; therefore C3d-K contains an additional 9 amino acids. It was concluded that the site responsible for causing leukocytosis may reside in the NH2-terminal nonapeptide portion of C3d-K and may represent the active center not only of C3d-K, but possibly C3e and L M F as well. We have synthesized the NH2-tesminal nonapeptide portion of C3d-K; the structure of this peptide is Thr-Leu-Asp-Pro-Glu-Arg-Leu-Gly-Arg (TLDPERLGR), and examined its biologic activity. At a concentration of 200 nmol/kg body weight, the nonapeptide and the des-Arg octapeptide ( T L D P E R L G ) induce leukocytosis in rabbits. The magnitude and time course of the response is like that of C3d-K and remarkably similar to that described for C3e. Tryptic scission of the nonapeptide into the corresponding tripeptide LGR and hexapeptide T L D P E R resulted in complete loss of activity indicating the leucyl and glycyl residues are important for function, even if the terminal arginine is not. The nonapeptide fails to suppress lymphocyte proliferation, and therefore we speculate that the immunomodulatory site exists elsewhere in the C3d-K molecule. Overall, the exact nature of C3b-derived factors that are immunoregulatory and have effects on circulating leukocytes was made considerably clearer with the biochemical characterization of C3d-K. Taken together with previous fragmentation studies a model for the C3 molecule can now be proposed. Indeed, this schematic representation has been upheld based on the recently completed c-DNA sequence of human C3 [16]. This model gives a more accurate structural basis from which to identify immunoregulatory fragments of C3 and localization of C3d-K helps to validate our concept that a single site in C3 may be responsible for the various activities assigned to iC3b, C3e and LMF.

4. D i s c u s s i o n

Clearly the immunoregulatory effects of the anaphylatoxins can be generated using both human and animal systems. For instance, human C3a is an effective agent on both human and murine lymphocytes. There are species differences, and quantitative variations in the immune responses elicited by a n a 212

phylatoxins from different animal sources do occur; however, the response itself remains qualitatively the same (Table 1). In terms of immunoregulation a more important consideration of the potential physiologic or pathophysiologic consequences of anaphylatoxins is the transient nature of these effector molecules in circulation. Under normal conditions the control mechanisms for the anaphylatoxins prevent a build-up of these factors. However, ongoing complement activation results in down-regulation of neutrophil activation and chemotactic function and may also promote immunoregulatory effects. The classical pathway of complement activation is an efficient mode for converting C3 and C4 but fails to generate C5a in significant quantities [17]. Therefore, primarily C3 factors are generated during classical pathway activation and the result would exert largely a suppressive influence on the immune response. This is perhaps a beneficial effect since the classical pathway is usually activated by immunoglobulins or immune complexes that may result from autoimmune diseases. In fact, C3a and other C3-derived suppressive factors could aid in modulating the autoimmune response. Conversely, the alternative pathway efficiently generates both C3a and C5a and the augmentation effects of C5a overrides the suppressive actions of C3a. Therefore, in host-defense mechanisms aimed at controlling external agents such as bacteria or yeast, enhancement of the immune response by C5a would be particularly beneficial to the host. Observations concerning involvement of complement factors in immunoregulation as indicated by in vitro assays, prompt further studies in two particular areas of investigation. First, it must be established that these same factors have some impact in vivo on immune responses. Numerous diseases are known that result in complement activation and lead to impairment in the immune response. An excellent example is acute malaria in children where the combination of C3 consumption [18] and immunosuppression [19] is documented. In vivo effects of complement on immune responses promises to be a most fruitful area for study having such tools now available as potent serum carboxypeptidase inhibitors, natural and synthetic effector molecules and complement-deficient laboratory animals. Secondly, the mechanism of action for anaphylatoxininduced sup-

pressive and a u g m e n t i n g effects are yet to be understood at the cellular level. T h e focus of further studies will be to identify cellular targets and unravel the m e c h a n i s m by which these c o m p l e m e n t mediators stimulate a chain of events resulting in their ultimate influence on the i m m u n e response.

Acknowledgements This w o r k was supported by N I H grants (l) C A 3 0 6 A and A I / C A 19723, C a r e e r D e v e l o p m e n t A w a r d CA00765 to ( E L M ) , (2) U S P H N a t i o n a l Research Service A w a r d AI06085 to ( M L T ) , and (3) HL25658 and AI17354 to ( T E H ) . W e wish to t h a n k N a n c y K a n t o r Baker for technical excellence.

References [1] Hugli, T. E. and Morgan, E. L. (1984) in: Contemporary Topics in Immunology (R. Snyderman, Ed.) pp. 109, Plenum Publ. Co,, New York. [2] Ross, G, D. and Newman, S, L, (1984) in: The Reticuloendothelial System: A Comprehensive Treatise (J. A. Bellanti and H. D. Herscowity, Eds.) pp. 87, Plenum Press, New York. [3] Weigle, W. O., Goodman, M. G., Morgan, E. L. and Hugli, T. E. (1983) Springer Sem. Immunopathol. 6, 173.

[4] Morgan, E. L., Weigle, W. O. and Hugli, T. E. (1982) J. Exp. Med. 155, 1412. [5] Hugli, T. E. (1981) in: Critical Reviews in Immunology, pp. 321, CRC Press Review, Boca Raton, Florida. [6] Morgan, E. L., Weigle, W. O., Erickson, B. W., Fok, K.-F. and Hugli, T. E. (1983) 3. lmmunol. 131, 2258. [7] Morgan, E. L., Thoman, M. L., Weigle, W. O. and Hugli, T. E. (1985)./. Immunol. 134, 51. [8] Payan, D. G., Trentham, D. E. and Goetzel, E. J. (1982) J. Exp. Med. 156, 756. [9] Morgan, E, L., Thoman, M. L,, Weigle, W. O. and Hugli, T. E. (1983)3. Immunol. 130, 1257. [10] Goodman, M. G., Chenoweth~ D. E. and Weigle, W. O. (1982) J. lmmunol. 129, 70. [11] Chenoweth, D. E., Lane, T. A., Rowe, J. G. and Hugli, T. E. (1980) Clin. lmmunol. Immunopathol. 15, 525. [12] Meuth, J. L., Morgan, E. L., DiScipio, R. G. and Hugli, T. E. (1983) J. Immunol. 130, 2605. [13] Thoman, M. L., Meuth, J. L., Morgan, E. L. and Hugli, T. E. (1984) .I. Immunol. 133, 2629. [14] Ghebrehiwet, B. and Muller-Eberhard, H. J. (1979) J. lmmunol. 123,616, [15] Rother, K. (1972) Eur. J. lmmunol. 2, 550. [16] de Bruijn, M. and Fey, G. (1985) Proc. Natl. Acad. Sci. U.S.A., in press. [17] Wagner, J. L. and Hugli, T. E. (1984) Analyt. Biochem. 136, 75. [18] Ade-Serrano, M. A., Ejezie, G. C. and Kassim, O. O. (1981) J. Clin. Microbiol. 13,195. [19] Williamson, W. A. and Greenwood, B. M. (1978) Lancet I, 1328.

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