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Glandular kallikrein in immunoregulation
New Foundationof Biology
225
Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Glandular Kallikrein in Immunoregulation
EDRIS SABBADINI 1, EVA NAGY 1, ALEXANDER FRED T. KISIL 1 and ISTVAN BERCZI 1
V(~RI~S 2, GERTRUDE VOROSOVA 3,
1Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada 2Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada 3Department of Anatomy, University of Manitoba, Winnipeg, Manitoba, Canada
ABSTRACT Glandular Kallikrein (GK) is an enzyme of the serine protease family capable of generating biologically active peptides by partially degrading various substrates. It is found in several tissues with particularly high concentrations in salivary glands, pancreas, kidney and the prostate gland. The physiological functions of this enzyme appear to vary according to the tissue in which it acts and the substrate(s) available in such tissues. Our interest in GK arose when we demonstrated that an immunoregulatory factor isolated from the salivary submandibular (SM) gland of rats, was rat GK (rGK). When added to cultures of lymphoid tissues, rGK induced a significant increase in lymphocyte proliferation manifested either in unstimulated cell cultures or in cultures of cells stimulated with suboptimal amounts of T cell mitogens. When injected subcutaneously, rGK induced a marked, albeit short lived, immunosuppressive effect in various experimental models including contact hyper-sensitivity to picryl chloride, allograft rejection, the production of antibody plaque-forming cells in the spleen of animals immunized with sheep red blood cells, and collagen-induced arthritis. All of the above effects were species-nonspecific inasmuch as murine GK and pig GK induced identical effects to rGK either in mice or in rats. Moreover, for these effects to take place, the enzymatic activity of GK had to be preserved. In the presence of inhibitors of proteolytic activity, both the in vivo and the in vitro immunoregulatory effects were abolished. This indicated that GK acts on a protein substrate to generate immunoregulatory peptides. If this is the case, the apparent conflict between in vitro stimulation and the suppression induced by subcutaneously injected GK can be explained as due to the accumulation of lymphocyte stimulating peptides in the in vitro cultures, while in vivo injection probably results in the rapid dispersal and degradation of such peptides leaving the organism depleted of substrate and unable to produce amounts of active peptides sufficient for a normal immune response. The effects described up to this point refer to in vitro phenomena or to the subcutaneous administration of GK. The fact that GK is found in high concentration in salivary glands and is secreted in saliva suggests that a significant physiological function of GK may occur following external salivary secretion. For this reason, we also tested the effect of orally administered GK in rats. Oral GK was shown to be required for the induction of tolerance to orally administered antigens. Rats subjected to the surgical removal of the SM gland (SMX) did
226
not develop tolerance to oral collagen given in doses that induced significant tolerance in normal controls. The oral administration of GK in the SMX animals fully restored the capacity to develop oral tolerance. This suggests that salivary GK is responsible for homeostasis in the mucosa associated lymphoid tissue which is responsible for the development of tolerance to oral antigens. The observation that GK may be used to enhance the induction of oral tolerance holds promises for the therapuetic application of oral GK in autoimmune diseases.
1.
INTRODUCTION
Kallikreins are a family of serine proteases capable of cleaving various substrates and generating biologically active peptides. In spite of the identity of names, tissue or glandular kallikreins should be distinguished from plasma kallikrein. They differ from plasma kallikrein in their genes of origin, molecular weight, amino acid sequences, substrates, peptide products and most probably physiological functions. There are at least 20 genes for tissue kallikrein in rodents [1], while in humans only 4 genes have been so far described. Of these, only one gene in each species codes for true glandular kallikrein. Of the rat genes at least 6-7 appear to be expressed in the submandibular (SM) gland [2]. These include true glandular kallikrein, tonin, c~ and ~, NGF, and the EGF-binding protein (EGF-BP), type A, B, and C. Here the term glandular kallikrein (GK) will apply to true GK only, while the general term kallikrein(s) will be used for any unspecified members of the tissue kallikrein family. True GK has been designated in various species as kallikrein-1 (K1) [3]. The best known substrates for GK action are hepatic-derived kininogens [3] which occur in two forms, low molecular weight kininogen (50 kDa) and high molecular weight kininogen (120 kDa). From the action of plasma kallikrein on high molecular weight kininogen a nonapeptide, bradikinin, is generated, while in most species GK gives rise to a decapeptide, kallidin (lys-bradikinin) from either low or high molecular weight kininogen. Kallidin is biologically active in itself but may also be further processed into bradikinin. An exception may be the GK of the rat SMG which was reported to produce bradikinin [4]. While the action of GK on kininogen is particularly well studied, the full range of GK substrates has not yet been investigated. Since GK is, and most of the times remains, localized in certain tissues, physiological substrates are likely to vary from tissue to tissue. Of particular interest here is the possibility that GK may activate or in some way regulate other immunologically active factors of the SM gland, including NGF, EGF/TGF~ and TGF[3. Thus, salivary gland GK may exert its immunological effects either via the production of classical kinins or via other immunologically active factors. Moreover, salivary GK is actively secreted in saliva [5] and would be expected to reach various points in the gastro-intestinal tract and act on various substrates there. Several in vitro effects of kallikreins on cells of the immune system have been reported. Thus, several authors have described mitogenic and co-mitogenic effects of kallikrein and other serine proteases. Such mitogenic effects were observed with thymocytes [6], T cells and B cells [7]. Although bradikinin may also have mitogenic effects [8], the involvement of this kinin in kallikrein-induced mitogenesis is not well investigated. Moreover, several proteases, including kallikrein, were shown to be involved in immunoglobulin isotype control. Thus Ishizaka described a kallikrein-like factor called glycosylation-enhancing factor, which induced CD4+ T cells to produce an IgE-potentiating factor and to favour the production of IgE by memory B cells [9]. Serine proteases from Schistosoma mansoni schistosomula were reported to enhance IgE production [10]. Moreover, the addition of kallikrein and other serine proteases in various
227
concentrations to cultures of B cells stimulated with LPS and IL 4 enhanced the production of IgE, IgG 1, or IgG3, depending on the enzyme concentration used [ 11 ]. Our interest in GK arose from studies on immunosuppressive factors in the SM gland of rats. The addition of crude extracts from rat SM glands to murine spleen and lymph node cultures stimulated with concanavalin A (Con A) induced either suppression (at high concentrations) or further stimulation (at lower concentrations) of proliferative activity [12]. This suggested that these extracts contained factors with suppressive effects as well as factors with the ability to enhance lymphocyte proliferation. Gel filtration of the crude extracts revealed that the in vitro suppressive activity was due to factors with molecular weight higher than 50 kilo Daltons (kDa), while stimulation was due to factors with molecular weight lower than 50 kDa (Figure 1). We tested the in vivo activity of both the higher and lower molecular weight fractions in the skin allograft, direct plaque forming cell response and in the delayed-type hypersensitivity (DTH) models [13]. As shown in Table I, and contrary to what one might have expected in view of their in vitro suppressive activity, the high molecular weight fractions did not have any significant effect in these models. On the other hand, the lower molecular weight fractions produced significant suppression in all three models. .I 6 0
-
120 =
,,7 o v
"(
80
ci.
40
!
!
o z's 8. 3 z- 8 0.93 0:3, o.,o Fresh hssue
(/J..g/ml)
Figure 1. Effects of pooled Sephacryl fractions of submandibular ( 0 , II) and parotid (O, F-I) glands on the Con A stimulated lymph node cell proliferation: ( 0 , 9
MW > 50 kDa; (m, D) MW < 50 kDa. Thesolid horizontal line is the
value of mean c.p.m, for the controls and the shaded area represents the 95% confidence limits. From Ref. 12.
Fractionation of the lower molecular weight pool of fractions through successive steps of hydrophobic interaction, anion exchange chromatography and finally gel filtration, led to the isolation a single protein (Figure 2) which retained the properties of in vitro stimulation of lymphocyte proliferation and in vivo immunosuppression (results not shown). The amino acid sequence of the isolated protein was determined [14] using an automated Edman degradation procedure [15]. Figure 3 shows the partial N-terminal amino acid sequence of the 40 kDa protein and of the members of the kallikrein family represented in the rat SMG. The x's (unidentified amino acids) in our sequence are probably cysteines which are destroyed in the Edman degradation process. If this is taken into account, the first 25 amino acids of our protein
228
has identical sequence
with that of true GK and differ from those of other members
of the
kallikrein family. In view of the fact that no rat genes with the same amino acid sequence are k n o w n , it w a s c o n c l u d e d t h a t t h e i s o l a t e d p r o t e i n w a s t r u e r a t G K ( r G K ) . Table I
Effects of high molecular weight (HMW) and low molecular weight (LMW) pools of gel filtration fractions in three in vivo immunological assays. Modified from Ref. 13.
Exp Model
Treatment a
Groups
Results ___SD b
Skin transplantation
10 daily doses
PBS
12.2 + 0.37
CBA/2J to C57B1/6J
Days 0 to 9
HMW
13.0 • 0.44
NS
LMW
14.7 _+0.76
p < 0.05
PBS
237.0 • 19.7
193.0 + 12.0
NS
Direct PFC
5 daily doses
Days 1 to 3
HMW
DTH (A/J mice)
2 daily doses
Days 4 and 6
HMW
LMW
119.6 • 10.0
PBS
19.0 • 0.70
18.2 + 0.49
NS
LMW
9.0 • 1.00
Significancec
p < 0.05
p < 0.01
a. The animals received the subcutaneous injection of 200 ~tl of PBS or PBS containing either the HMW or the LMW fractions. The doses corresponded to one-half of a SM gland (0.965 mg or 0.53 mg, respectively) and to one gland in the other models (1.93 mg or 1.06 mg, respectively). b. Skin transplantation results are expressed as mean survival time; PFC response is expressed as the number of IgM anti-SRBC PFC per one million splenocytes; DTH results are expressed as increases of ear thickness in 1/10 mm units 24 hours after challenge. c. Significance was determined by two-sample t-test, using the PBS-treated group as the control; NS, not significant.
. . . .
97.4 kD 66.2 kD
I~
42.7 kD
~.
31kD
~,
21.5 kD
I~
i 84184184 i 51 i.
14.4 kD
1
2
Figure 2. SDS-PAGE of the purified protein from rat SM gland (lane 2). Molecular weight standards are shown in lane 1. From Ref. 14.
229
Protein
40
1
i0
15
20
25
WGGYNxEMNSQPWQVAVYYFGEYLx
kDa
Gland.
5
Kallikrein
VVGGYNCEMNSQPWQVAVYYFGEYLC
GYNy~ ~ NSQPWQVA~
KLP-S3
K sQpwQvA
Tonin Antigen 7
:I VGG'i K rd~ Kt~SQPWQV'~
T-kininogenase
I VGG~ K ZE K NSQPWQV~
Proteinase
I
B
Proteinase A Figure 3. Partial N-terminal amino acid of the 40 kDa protein isolated from rat SM gland compared with those of members of kallikrein family expressed in the rat SM gland. The boxed areas represent regions of identity with the 40 kDa protein. Blank spaces are used to align homologous sequences in different proteins, x = not identified. From Ref. 14.
The esterase activity of the isolated rGK was approximately the same as that of a commercially obtained porcine GK (pGK) when measured in the 2-N-benzoyl-arginine ethyl estez (BAEE) assay [16]. Different concentrations of aprotinin induced different degrees of inhibition. Figure 4 demonstrates the effects of rGK and pGK in the presence or in the absence of aprotinin on the proliferative activity of Con A stimulated murine lymph node cells. The same concentrations of pGK induced similar co-mitogenic effects. The Con A concentration used in these experiments was such as to induce only suboptimal mitogenic effects, suitable for the demonstration of the co-mitogenic activity of rGK. The addition of different concentrations of aprotinin to the co-stimulated cultures induced dose dependent suppression. It should be noted that the highest concentration of aprotinin used in this experiment (1.5 ~tg/ml or 6 ~tg/culture) was capable of inducing some 90% inhibition in the B AEE assay. On the other hand, the lowest concentration of aprotinin (1.5 ~tg/culture) induced approximately 40% inhibition of the enzymatic activity and partial inhibition of the co-mitogenic activity. The results of an in vivo experiment performed along the same lines are presented in Figure 5. A DTH reaction was induced in mice sensitized with picryl chloride and challenged with the same agent six days later. The injection of rGK 24 hour before challenge resulted in nearly complete (~ 87%) suppression of the response. Similar suppression was obtained with pGK (-72%). The dose of rGK used in this experiment (57 ~tg/animal) was based on our previous experience. The higher of the two aprotinin doses (190 ~tg/animal) was selected so as to provide, after dilution in the blood stream, a concentration similar to that used in the in vitro experiments. The suppressive effects of rGK and pGK were almost completely removed by the injection of this higher dose of aprotinin given immediately before GK injection (inhibition nearly -8%). On the other hand, the lower dose (95 ~tg) of aprotinin induced only partial suppression of the rGK effects (inhibition still -60%). These two experiments clearly demonstrate that
230
the enzymatic activity of GK must be preserved in order to retain its in vivo and i n vitro immunological effects.
cpm xl,000 10 15 I
20
I
!
PBS Ill
rGK
II1|
II
I
-t-
I
§
Apr. (6 lag) rGK + Apr. (6 I~g) rGK + Apr. (3 I~g)
-q-
rGK + Apr. (1.5 I~g)
II
III
I
I
I
pGK
I
I
I
-t_ff
pGK + Apr. (6 Ixg)
Figure 4. Increased proliferative activity of Con A stimulated A/J lymph node cells induced by rGK and pGK and reversal of this effect with aprotinin. The rGK and pGK doses were 1.78 g/culture (final concentration 0.22 ~tM/1). Aprotinin (Apr.) doses were: 6 gg, 3 gg, or 1.5 gg per culture 4.6 M/i, 2.3 M/l, and 1.15 M/l, respectively). Results are expressed as counts per minute (C.P.M.) in triplicate cultures (+S.D.). From Ref. 14.
A Ear thickness ~rn rn) 0.5 1.0 I
I
+
PBS
+
rGK Apr. (190 I~g) rGK + Apr. (190 I~g)
II
el
rGK + Apr. (95 I~g) pGK pGK + Apr. (190 Jag)
II
, I
, , II
I
"t-
II
-I-
Figure 5. Effects of rGK and pGK on the DTH response of A/J mice immunized with picryl chloride and reversal of such effects with aprotinin. Aprotinin (Apr.) was injected subcutaneously as a full dose (190 pg per animal) or as a half dose (95 ~tg per animal). Fifteen minutes later the animals received a further subcutaneous injection of rGK or pGK in 0.2 ml of PBS or PBS only. Results are expressed in terms of the increase of the thickness of the challenged ear over the pre-challenge values. Unimmunized controls (not shown) did not show any increases of the ear thickness. From Ref. 14.
231
Table II demonstrates the effects of varying the time of GK injection with respect to the time of immunization or of challenge in the DTH model in mice. If given before immunization, GK had a suppressive effect lasting for at least fourteen days. This suggested that the animals did not develop any immunity when presented with the antigen. On the other hand, if GK was given after the development of immunity, it induced a short lived suppression of the skin reaction with a full recovery of reactivity a week after GK administration. This demonstrated that GK did not affect the state of immunity of an animal and suppressed the skin reaction with a mechanism that may be either immunological or anti-inflammatory. It should be noted that the half life of GK is such that 24 hours after injection, i.e. at the time of antigen administration for either immnunization or challenge, none of the injected GK is present in the animal. Thus, GK mediated inhibition probably occurs indirectly, following production of mediators dependent on GK enzymatic action. Table II
Effects of the intradermal injection o f r G K given before or after immunization in the DTH model.
Treatment a
None
Test on day: b
7
14
18.4 + 0.76
17.6 + 0.92
rGK, Day-1
6.0 + 1.2"
7.6 + 0,98*
rGK, Day+6
4.4 + 0.63*
16.8 + 1.12
rGK, Day+13
19.0 + 1.24
5.6 + 0.78*
a. A/J mice were immunized by the application of 0.1 ml of a 5% solution of picryl chloride in ethanol to the skin of the abdomen; the day of immunization is referred to as day 0; the animals a single dose of 60 ~tg of rGK in 200 ~tl of PBS on the days indicated. b. Animals were tested with the application of a 1% solution of picryl chloride in olive oil on one ear. Results are expressed in terms of the increase if the challenged ear over the pre-challenge values using units equivalent to 1/10 ram. * p < 0.05.
Figure 6 shows the results of an experiment in the collagen arthritis (CA) model in rats. A single injection of GK given at the time when the arthritic reaction begins to flare induced an almost complete suppression which lasted 4-5 days, followed by the return to central levels of arthritis. In contrast, repeated injections maintained suppression for the entire duration of the experiment. Thus, this experiment confirmed that the effects of GK in immune animals are short lived and do not reduce the state of immunity of the animals. The effects described up to this point refer to in vitro phenomena or to the subcutaneous administration of GK. The fact that GK is found in high concentration in salivary glands and is secreted in saliva suggests that a significant physiological function of GK may occur following external salivary secretion. For this reason, we tested the effects of orally administered GK in rats. Experiments of this nature were carried out in rats using the collagen arthritis model. This involves the injection of Type II collagen in an oil based adjuvant which induces an arthritic reaction beginning two weeks after immunization, which lasts for the next 3-4 weeks.
232
10 x --
8 6
9 w,,,,,i
4
"
12
i
9
u
9
18
24
30
36
Days Figure 6. Effects of a single (V, day 14) or multiple ( I , days 14, 18, and 24) injections of a semi-purified preparation of rGK in the CA model. Controls (O) received PBS only.
The variables we tested included the effects of a pre-treatment with oral collagen to induce tolerance to the subsequent immunization; the effects of the surgical removal of the submandibular gland; and the effect of oral administration of GK, given before tolerization and/or before immunization to normal and sialoadenectomized rats. Figure 6 demonstrates the results of one of such experiments. The oral pre-treatment with Type II collagen significantly reduced the arthritic response in normal rats but had no such effect in sialoadenectomized animals. In contrast, the oral administration of GK significantly reduced the arthritic reaction. Figure 7 shows the results of another experiment which confirmed that sialoadenectomy interferes with tolerance induction. Moreover, this experiment demonstrated that the oral administration of GK to sialoadenectomized rats restores the ability of the mucosal immune system of these animals to develop tolerance upon oral collagen administration. These results point to similarities and differences in the action of GK depending on whether it is used in vitro or in vivo, and whether it is injected or given orally. The most prominent in vitro effect was to induce stimulation of lymphocyte proliferation, while the most prominent in vivo effect was an immunosuppressive one. One hypothesis to explain this apparent discrepancy in vivo suggests that immune deviation occurs following administration of GK in vivo, involving a reduction in the responses we assessed, but a stimulation of other responses which we did not resume. One example of such an immune deviation would be an altered balance of TH1 to TH2 responses with a decrease of TH1 activity. The responses we found to be suppressed by GK treatment were cell- mediated ones (DTH, allograft rejection and CA) or T-dependent IgM production (direct PFC response). These responses would be suppressed by any mechanisms that reduce TH1 activity or favor the switch from IgM to any other immunoglobulin class. This explanation is consistent with the observation that oral GK favors the induction of oral tolerance, a reaction thought to involve a deviation from cell-mediated responses to IgA production and induced by increased activity of TGF- (and IL-4) producing
233
4.5
A
IE IE nr"
uJ I--
4.0
I..U
< a
3.5
o lo
2'0 DAYS AFTER IMMUNIZATION
Figure 7. Effects of SMX and semi-purified oral rGK on the arthritic response and on the induction of oral tolerance in Sprague Dawley rats. (O) immunized controls; (9 oral collagen followed by immunization; (A) SMX followed by immunization; (m) SMX, oral collagen, followed by immunization; (@) oral rGK, followed by immunization.
TH3_ type cells. Alternately suppressor cells may be activated by GK. Yet another possible explanation for the apparent discrepancy of in vitro versus in vivo effects would suggest that in vitro stimulation of lymphocyte proliferation was due to the formation of stimulatory peptides, formed by the action of GK on some substrate(s) contained in the foetal calf serum present in our cultures. Under in vivo conditions, the proteolytic action of GK may result in decreased concentration of the same substrate(s) and in a deficient production of stimulatory peptides for a few days, until the substrates are reconstituted in full. Differences in GK action were also observed when the route of GK administration changed from subcutaneous to oral. In both situations, some suppression of immune reactions was observed if the treatment was simultaneous with or shortly preceded, antigen administration. On the other hand, the oral administration of GK appeared to enhance the induction of tolerance if given together with the oral antigen. The ensuing suppression of the arthritic reaction exceeded in duration and in magnitude the "immunosuppressive" effect of GK alone, administered either by injection or by mouth. The same experiments also demonstrated that oral tolerance could not be induced by antigen alone in sialoadenectomized animals. Oral tolerance is a state of antigen specific hyporesponsiveness which follows oral delivery of an antigen. It represents a protective reaction by the gut associated mucosal tissue (GALT) to prevent unnecessary and potentially harmful reactions to food antigens. It involves more than one mechanism. High antigen doses induce clonal deletion, while lower doses induce an active form of suppression or immune deviation, mediated by TGF- and IL-4 producing T cells,
234
6.5
6.0
E E v nLU i'LU
5.5
< a
.,,..=
n<<:: 5.0
4.5 4.3
..j/; 0 14 1'61'8 2'1'2'3;~5 28 31
,g :35:36
4'2 Days
DAYS AFTER IMMUNIZATION Figure 8. Effects of SMX and oral pGK on the induction of oral tolerance in Lewis rats. (0) immunized controls; (O) oral collagen followed by immunization; (A) SMX followed by immunization; (I) SMX, oral collagen, followed by immunization; (I-]) SMX, oral collagen, oral pGK, followed by immunization.
referred to by some authors as TH3 cells. The action of these cells suppresses TH1 responses and favors TH2 responses [17-19]. From Peyer's Patches, these regulatory cells migrate to periferal lymphoid organs rendering systemic the modified responsiveness. Thus, our results suggest that the SM gland plays a significant role in maintaining the normal responsiveness of GALT and that GK secretion in saliva is one of the factors involved in this function. The immunological properties of GK make this molecule an interesting candidate in the treatment of autoimmune diseases.
ACKNOWLEDGMENTS The experiments described here were carried out in collaboration with A. Kemp, L. Mellow, M. Abdelhaleem (experiments with crude SM gland extracts and with semi-purified rGK). Financial support was from the Medical Reasearch Council of Canada.
235
REFERENCES
.
.
.
.
.
.
10.
11. 12. 13. 14. 15. 16. 17.
18. 19.
Fuller PJ, Funder JW. The cellular physiology of glandular kallikrein. Kidney 1986; 29: 953. Wines DR, Brady JM, Pritchett DB, Roberts JL, MacDonald RJ. Organization and expression of the rat kallikrein gene family. J Biol Chem 1989; 264: 7653. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 1992; 44: 1. Alhenc-Gelas F, Marchetti J, Allegrini J, Corvol P, Menanrd J. Measurement of urinary kallikrein activity species differences in kinin production. Biochim Biophys. Acta 1981; 677: 477. Simpson JAV, Chao J, Chao L. Localization of kallikrein gene family proteases in rat tissues. Agents and Actions-Suppl 1992; 38 (Pt. 1): 595. Naughton MA, Geczy C, Bender V, Hoffman H, Hamilton E. Esteropeptidase and thymotropic activity of a protein isolated from the mouse submaxillary gland. B iochim Biophys Acta 1972; 263: 106. Hu ZQ, Murakami K, Ikigai H, Shimamura T. Enhancement of lymphocyte proliferation by mouse glandular kallikrein. Immunol Lett 1992; 32: 85. Perris AD, Whitfield JF. The mitogenic action of bradykinin on thymic lymphocytes and its dependence on calcium. Proc Soc Exp Biol Med 1969; 130: 1198. Ishizaka K. Twenty years with IgE: from the identification of IgE to regulatory factors for the IgE response. J Immunol 1985; 135: i-x. Verwaerde C, Auriault C, Neyrinck JL, Capron A. Properties of serine proteases of Schistosoma mansoni. Shistosomula involved in the regulation of IgE synthesis. Scand J Immunol 1988; 27: 17. Matsushita S, Katz DH. B iphasic effect of kallikrein on IgE and IgG1 syntheses by LPS/IL4 stimulated B cells. Cell Immunol 1993; 146: 210. Kemp A, Mellow L, Sabbadini E. Suppression and enhancement of in vitro lymphocyte reactivity by factors in rat submandibular gland extracts. Immunology 1985; 56: 261. Abdelhaleem M, Sabbadini E. Identification of immunosuppressive fractions from the rat submandibular salivary gland. Immunology 1992; 76: 331. Nagy E, Berczi I, Sabbadini E. Immunoregulatory effects of glandular kallikrein from the salivary submandibular gland of rats. Neuroimmunomodulation 1997; 4: 107. Edman P, Begg G. A protein sequenator. Eur J B iochem 1967; 1: 80. Trautschold I. Assay methods in the kinin system. In Erdos G: Handbook of experimental pharmacology, Bradikinin, kallidin, and kallikrein. Berlin, Springer, 1970; 25: 52-81. Weiner HL, Friedman A, Miller A, et al. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol 1994; 12: 809. Weiner HL. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997; 18: 335. Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci 1994; 91: 6688.
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Glandular Kallikrein in Immunoregulation
EDRIS SABBADINI 1, EVA NAGY 1, ALEXANDER FRED T. KISIL 1 and ISTVAN BERCZI 1
V(~RI~S 2, GERTRUDE VOROSOVA 3,
1Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada 2Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada 3Department of Anatomy, University of Manitoba, Winnipeg, Manitoba, Canada
ABSTRACT Glandular Kallikrein (GK) is an enzyme of the serine protease family capable of generating biologically active peptides by partially degrading various substrates. It is found in several tissues with particularly high concentrations in salivary glands, pancreas, kidney and the prostate gland. The physiological functions of this enzyme appear to vary according to the tissue in which it acts and the substrate(s) available in such tissues. Our interest in GK arose when we demonstrated that an immunoregulatory factor isolated from the salivary submandibular (SM) gland of rats, was rat GK (rGK). When added to cultures of lymphoid tissues, rGK induced a significant increase in lymphocyte proliferation manifested either in unstimulated cell cultures or in cultures of cells stimulated with suboptimal amounts of T cell mitogens. When injected subcutaneously, rGK induced a marked, albeit short lived, immunosuppressive effect in various experimental models including contact hyper-sensitivity to picryl chloride, allograft rejection, the production of antibody plaque-forming cells in the spleen of animals immunized with sheep red blood cells, and collagen-induced arthritis. All of the above effects were species-nonspecific inasmuch as murine GK and pig GK induced identical effects to rGK either in mice or in rats. Moreover, for these effects to take place, the enzymatic activity of GK had to be preserved. In the presence of inhibitors of proteolytic activity, both the in vivo and the in vitro immunoregulatory effects were abolished. This indicated that GK acts on a protein substrate to generate immunoregulatory peptides. If this is the case, the apparent conflict between in vitro stimulation and the suppression induced by subcutaneously injected GK can be explained as due to the accumulation of lymphocyte stimulating peptides in the in vitro cultures, while in vivo injection probably results in the rapid dispersal and degradation of such peptides leaving the organism depleted of substrate and unable to produce amounts of active peptides sufficient for a normal immune response. The effects described up to this point refer to in vitro phenomena or to the subcutaneous administration of GK. The fact that GK is found in high concentration in salivary glands and is secreted in saliva suggests that a significant physiological function of GK may occur following external salivary secretion. For this reason, we also tested the effect of orally administered GK in rats. Oral GK was shown to be required for the induction of tolerance to orally administered antigens. Rats subjected to the surgical removal of the SM gland (SMX) did
226
not develop tolerance to oral collagen given in doses that induced significant tolerance in normal controls. The oral administration of GK in the SMX animals fully restored the capacity to develop oral tolerance. This suggests that salivary GK is responsible for homeostasis in the mucosa associated lymphoid tissue which is responsible for the development of tolerance to oral antigens. The observation that GK may be used to enhance the induction of oral tolerance holds promises for the therapuetic application of oral GK in autoimmune diseases.
1.
INTRODUCTION
Kallikreins are a family of serine proteases capable of cleaving various substrates and generating biologically active peptides. In spite of the identity of names, tissue or glandular kallikreins should be distinguished from plasma kallikrein. They differ from plasma kallikrein in their genes of origin, molecular weight, amino acid sequences, substrates, peptide products and most probably physiological functions. There are at least 20 genes for tissue kallikrein in rodents [1], while in humans only 4 genes have been so far described. Of these, only one gene in each species codes for true glandular kallikrein. Of the rat genes at least 6-7 appear to be expressed in the submandibular (SM) gland [2]. These include true glandular kallikrein, tonin, c~ and ~, NGF, and the EGF-binding protein (EGF-BP), type A, B, and C. Here the term glandular kallikrein (GK) will apply to true GK only, while the general term kallikrein(s) will be used for any unspecified members of the tissue kallikrein family. True GK has been designated in various species as kallikrein-1 (K1) [3]. The best known substrates for GK action are hepatic-derived kininogens [3] which occur in two forms, low molecular weight kininogen (50 kDa) and high molecular weight kininogen (120 kDa). From the action of plasma kallikrein on high molecular weight kininogen a nonapeptide, bradikinin, is generated, while in most species GK gives rise to a decapeptide, kallidin (lys-bradikinin) from either low or high molecular weight kininogen. Kallidin is biologically active in itself but may also be further processed into bradikinin. An exception may be the GK of the rat SMG which was reported to produce bradikinin [4]. While the action of GK on kininogen is particularly well studied, the full range of GK substrates has not yet been investigated. Since GK is, and most of the times remains, localized in certain tissues, physiological substrates are likely to vary from tissue to tissue. Of particular interest here is the possibility that GK may activate or in some way regulate other immunologically active factors of the SM gland, including NGF, EGF/TGF~ and TGF[3. Thus, salivary gland GK may exert its immunological effects either via the production of classical kinins or via other immunologically active factors. Moreover, salivary GK is actively secreted in saliva [5] and would be expected to reach various points in the gastro-intestinal tract and act on various substrates there. Several in vitro effects of kallikreins on cells of the immune system have been reported. Thus, several authors have described mitogenic and co-mitogenic effects of kallikrein and other serine proteases. Such mitogenic effects were observed with thymocytes [6], T cells and B cells [7]. Although bradikinin may also have mitogenic effects [8], the involvement of this kinin in kallikrein-induced mitogenesis is not well investigated. Moreover, several proteases, including kallikrein, were shown to be involved in immunoglobulin isotype control. Thus Ishizaka described a kallikrein-like factor called glycosylation-enhancing factor, which induced CD4+ T cells to produce an IgE-potentiating factor and to favour the production of IgE by memory B cells [9]. Serine proteases from Schistosoma mansoni schistosomula were reported to enhance IgE production [10]. Moreover, the addition of kallikrein and other serine proteases in various
227
concentrations to cultures of B cells stimulated with LPS and IL 4 enhanced the production of IgE, IgG 1, or IgG3, depending on the enzyme concentration used [ 11 ]. Our interest in GK arose from studies on immunosuppressive factors in the SM gland of rats. The addition of crude extracts from rat SM glands to murine spleen and lymph node cultures stimulated with concanavalin A (Con A) induced either suppression (at high concentrations) or further stimulation (at lower concentrations) of proliferative activity [12]. This suggested that these extracts contained factors with suppressive effects as well as factors with the ability to enhance lymphocyte proliferation. Gel filtration of the crude extracts revealed that the in vitro suppressive activity was due to factors with molecular weight higher than 50 kilo Daltons (kDa), while stimulation was due to factors with molecular weight lower than 50 kDa (Figure 1). We tested the in vivo activity of both the higher and lower molecular weight fractions in the skin allograft, direct plaque forming cell response and in the delayed-type hypersensitivity (DTH) models [13]. As shown in Table I, and contrary to what one might have expected in view of their in vitro suppressive activity, the high molecular weight fractions did not have any significant effect in these models. On the other hand, the lower molecular weight fractions produced significant suppression in all three models. .I 6 0
-
120 =
,,7 o v
"(
80
ci.
40
!
!
o z's 8. 3 z- 8 0.93 0:3, o.,o Fresh hssue
(/J..g/ml)
Figure 1. Effects of pooled Sephacryl fractions of submandibular ( 0 , II) and parotid (O, F-I) glands on the Con A stimulated lymph node cell proliferation: ( 0 , 9
MW > 50 kDa; (m, D) MW < 50 kDa. Thesolid horizontal line is the
value of mean c.p.m, for the controls and the shaded area represents the 95% confidence limits. From Ref. 12.
Fractionation of the lower molecular weight pool of fractions through successive steps of hydrophobic interaction, anion exchange chromatography and finally gel filtration, led to the isolation a single protein (Figure 2) which retained the properties of in vitro stimulation of lymphocyte proliferation and in vivo immunosuppression (results not shown). The amino acid sequence of the isolated protein was determined [14] using an automated Edman degradation procedure [15]. Figure 3 shows the partial N-terminal amino acid sequence of the 40 kDa protein and of the members of the kallikrein family represented in the rat SMG. The x's (unidentified amino acids) in our sequence are probably cysteines which are destroyed in the Edman degradation process. If this is taken into account, the first 25 amino acids of our protein
228
has identical sequence
with that of true GK and differ from those of other members
of the
kallikrein family. In view of the fact that no rat genes with the same amino acid sequence are k n o w n , it w a s c o n c l u d e d t h a t t h e i s o l a t e d p r o t e i n w a s t r u e r a t G K ( r G K ) . Table I
Effects of high molecular weight (HMW) and low molecular weight (LMW) pools of gel filtration fractions in three in vivo immunological assays. Modified from Ref. 13.
Exp Model
Treatment a
Groups
Results ___SD b
Skin transplantation
10 daily doses
PBS
12.2 + 0.37
CBA/2J to C57B1/6J
Days 0 to 9
HMW
13.0 • 0.44
NS
LMW
14.7 _+0.76
p < 0.05
PBS
237.0 • 19.7
193.0 + 12.0
NS
Direct PFC
5 daily doses
Days 1 to 3
HMW
DTH (A/J mice)
2 daily doses
Days 4 and 6
HMW
LMW
119.6 • 10.0
PBS
19.0 • 0.70
18.2 + 0.49
NS
LMW
9.0 • 1.00
Significancec
p < 0.05
p < 0.01
a. The animals received the subcutaneous injection of 200 ~tl of PBS or PBS containing either the HMW or the LMW fractions. The doses corresponded to one-half of a SM gland (0.965 mg or 0.53 mg, respectively) and to one gland in the other models (1.93 mg or 1.06 mg, respectively). b. Skin transplantation results are expressed as mean survival time; PFC response is expressed as the number of IgM anti-SRBC PFC per one million splenocytes; DTH results are expressed as increases of ear thickness in 1/10 mm units 24 hours after challenge. c. Significance was determined by two-sample t-test, using the PBS-treated group as the control; NS, not significant.
. . . .
97.4 kD 66.2 kD
I~
42.7 kD
~.
31kD
~,
21.5 kD
I~
i 84184184 i 51 i.
14.4 kD
1
2
Figure 2. SDS-PAGE of the purified protein from rat SM gland (lane 2). Molecular weight standards are shown in lane 1. From Ref. 14.
229
Protein
40
1
i0
15
20
25
WGGYNxEMNSQPWQVAVYYFGEYLx
kDa
Gland.
5
Kallikrein
VVGGYNCEMNSQPWQVAVYYFGEYLC
GYNy~ ~ NSQPWQVA~
KLP-S3
K sQpwQvA
Tonin Antigen 7
:I VGG'i K rd~ Kt~SQPWQV'~
T-kininogenase
I VGG~ K ZE K NSQPWQV~
Proteinase
I
B
Proteinase A Figure 3. Partial N-terminal amino acid of the 40 kDa protein isolated from rat SM gland compared with those of members of kallikrein family expressed in the rat SM gland. The boxed areas represent regions of identity with the 40 kDa protein. Blank spaces are used to align homologous sequences in different proteins, x = not identified. From Ref. 14.
The esterase activity of the isolated rGK was approximately the same as that of a commercially obtained porcine GK (pGK) when measured in the 2-N-benzoyl-arginine ethyl estez (BAEE) assay [16]. Different concentrations of aprotinin induced different degrees of inhibition. Figure 4 demonstrates the effects of rGK and pGK in the presence or in the absence of aprotinin on the proliferative activity of Con A stimulated murine lymph node cells. The same concentrations of pGK induced similar co-mitogenic effects. The Con A concentration used in these experiments was such as to induce only suboptimal mitogenic effects, suitable for the demonstration of the co-mitogenic activity of rGK. The addition of different concentrations of aprotinin to the co-stimulated cultures induced dose dependent suppression. It should be noted that the highest concentration of aprotinin used in this experiment (1.5 ~tg/ml or 6 ~tg/culture) was capable of inducing some 90% inhibition in the B AEE assay. On the other hand, the lowest concentration of aprotinin (1.5 ~tg/culture) induced approximately 40% inhibition of the enzymatic activity and partial inhibition of the co-mitogenic activity. The results of an in vivo experiment performed along the same lines are presented in Figure 5. A DTH reaction was induced in mice sensitized with picryl chloride and challenged with the same agent six days later. The injection of rGK 24 hour before challenge resulted in nearly complete (~ 87%) suppression of the response. Similar suppression was obtained with pGK (-72%). The dose of rGK used in this experiment (57 ~tg/animal) was based on our previous experience. The higher of the two aprotinin doses (190 ~tg/animal) was selected so as to provide, after dilution in the blood stream, a concentration similar to that used in the in vitro experiments. The suppressive effects of rGK and pGK were almost completely removed by the injection of this higher dose of aprotinin given immediately before GK injection (inhibition nearly -8%). On the other hand, the lower dose (95 ~tg) of aprotinin induced only partial suppression of the rGK effects (inhibition still -60%). These two experiments clearly demonstrate that
230
the enzymatic activity of GK must be preserved in order to retain its in vivo and i n vitro immunological effects.
cpm xl,000 10 15 I
20
I
!
PBS Ill
rGK
II1|
II
I
-t-
I
§
Apr. (6 lag) rGK + Apr. (6 I~g) rGK + Apr. (3 I~g)
-q-
rGK + Apr. (1.5 I~g)
II
III
I
I
I
pGK
I
I
I
-t_ff
pGK + Apr. (6 Ixg)
Figure 4. Increased proliferative activity of Con A stimulated A/J lymph node cells induced by rGK and pGK and reversal of this effect with aprotinin. The rGK and pGK doses were 1.78 g/culture (final concentration 0.22 ~tM/1). Aprotinin (Apr.) doses were: 6 gg, 3 gg, or 1.5 gg per culture 4.6 M/i, 2.3 M/l, and 1.15 M/l, respectively). Results are expressed as counts per minute (C.P.M.) in triplicate cultures (+S.D.). From Ref. 14.
A Ear thickness ~rn rn) 0.5 1.0 I
I
+
PBS
+
rGK Apr. (190 I~g) rGK + Apr. (190 I~g)
II
el
rGK + Apr. (95 I~g) pGK pGK + Apr. (190 Jag)
II
, I
, , II
I
"t-
II
-I-
Figure 5. Effects of rGK and pGK on the DTH response of A/J mice immunized with picryl chloride and reversal of such effects with aprotinin. Aprotinin (Apr.) was injected subcutaneously as a full dose (190 pg per animal) or as a half dose (95 ~tg per animal). Fifteen minutes later the animals received a further subcutaneous injection of rGK or pGK in 0.2 ml of PBS or PBS only. Results are expressed in terms of the increase of the thickness of the challenged ear over the pre-challenge values. Unimmunized controls (not shown) did not show any increases of the ear thickness. From Ref. 14.
231
Table II demonstrates the effects of varying the time of GK injection with respect to the time of immunization or of challenge in the DTH model in mice. If given before immunization, GK had a suppressive effect lasting for at least fourteen days. This suggested that the animals did not develop any immunity when presented with the antigen. On the other hand, if GK was given after the development of immunity, it induced a short lived suppression of the skin reaction with a full recovery of reactivity a week after GK administration. This demonstrated that GK did not affect the state of immunity of an animal and suppressed the skin reaction with a mechanism that may be either immunological or anti-inflammatory. It should be noted that the half life of GK is such that 24 hours after injection, i.e. at the time of antigen administration for either immnunization or challenge, none of the injected GK is present in the animal. Thus, GK mediated inhibition probably occurs indirectly, following production of mediators dependent on GK enzymatic action. Table II
Effects of the intradermal injection o f r G K given before or after immunization in the DTH model.
Treatment a
None
Test on day: b
7
14
18.4 + 0.76
17.6 + 0.92
rGK, Day-1
6.0 + 1.2"
7.6 + 0,98*
rGK, Day+6
4.4 + 0.63*
16.8 + 1.12
rGK, Day+13
19.0 + 1.24
5.6 + 0.78*
a. A/J mice were immunized by the application of 0.1 ml of a 5% solution of picryl chloride in ethanol to the skin of the abdomen; the day of immunization is referred to as day 0; the animals a single dose of 60 ~tg of rGK in 200 ~tl of PBS on the days indicated. b. Animals were tested with the application of a 1% solution of picryl chloride in olive oil on one ear. Results are expressed in terms of the increase if the challenged ear over the pre-challenge values using units equivalent to 1/10 ram. * p < 0.05.
Figure 6 shows the results of an experiment in the collagen arthritis (CA) model in rats. A single injection of GK given at the time when the arthritic reaction begins to flare induced an almost complete suppression which lasted 4-5 days, followed by the return to central levels of arthritis. In contrast, repeated injections maintained suppression for the entire duration of the experiment. Thus, this experiment confirmed that the effects of GK in immune animals are short lived and do not reduce the state of immunity of the animals. The effects described up to this point refer to in vitro phenomena or to the subcutaneous administration of GK. The fact that GK is found in high concentration in salivary glands and is secreted in saliva suggests that a significant physiological function of GK may occur following external salivary secretion. For this reason, we tested the effects of orally administered GK in rats. Experiments of this nature were carried out in rats using the collagen arthritis model. This involves the injection of Type II collagen in an oil based adjuvant which induces an arthritic reaction beginning two weeks after immunization, which lasts for the next 3-4 weeks.
232
10 x --
8 6
9 w,,,,,i
4
"
12
i
9
u
9
18
24
30
36
Days Figure 6. Effects of a single (V, day 14) or multiple ( I , days 14, 18, and 24) injections of a semi-purified preparation of rGK in the CA model. Controls (O) received PBS only.
The variables we tested included the effects of a pre-treatment with oral collagen to induce tolerance to the subsequent immunization; the effects of the surgical removal of the submandibular gland; and the effect of oral administration of GK, given before tolerization and/or before immunization to normal and sialoadenectomized rats. Figure 6 demonstrates the results of one of such experiments. The oral pre-treatment with Type II collagen significantly reduced the arthritic response in normal rats but had no such effect in sialoadenectomized animals. In contrast, the oral administration of GK significantly reduced the arthritic reaction. Figure 7 shows the results of another experiment which confirmed that sialoadenectomy interferes with tolerance induction. Moreover, this experiment demonstrated that the oral administration of GK to sialoadenectomized rats restores the ability of the mucosal immune system of these animals to develop tolerance upon oral collagen administration. These results point to similarities and differences in the action of GK depending on whether it is used in vitro or in vivo, and whether it is injected or given orally. The most prominent in vitro effect was to induce stimulation of lymphocyte proliferation, while the most prominent in vivo effect was an immunosuppressive one. One hypothesis to explain this apparent discrepancy in vivo suggests that immune deviation occurs following administration of GK in vivo, involving a reduction in the responses we assessed, but a stimulation of other responses which we did not resume. One example of such an immune deviation would be an altered balance of TH1 to TH2 responses with a decrease of TH1 activity. The responses we found to be suppressed by GK treatment were cell- mediated ones (DTH, allograft rejection and CA) or T-dependent IgM production (direct PFC response). These responses would be suppressed by any mechanisms that reduce TH1 activity or favor the switch from IgM to any other immunoglobulin class. This explanation is consistent with the observation that oral GK favors the induction of oral tolerance, a reaction thought to involve a deviation from cell-mediated responses to IgA production and induced by increased activity of TGF- (and IL-4) producing
233
4.5
A
IE IE nr"
uJ I--
4.0
I..U
< a
3.5
o lo
2'0 DAYS AFTER IMMUNIZATION
Figure 7. Effects of SMX and semi-purified oral rGK on the arthritic response and on the induction of oral tolerance in Sprague Dawley rats. (O) immunized controls; (9 oral collagen followed by immunization; (A) SMX followed by immunization; (m) SMX, oral collagen, followed by immunization; (@) oral rGK, followed by immunization.
TH3_ type cells. Alternately suppressor cells may be activated by GK. Yet another possible explanation for the apparent discrepancy of in vitro versus in vivo effects would suggest that in vitro stimulation of lymphocyte proliferation was due to the formation of stimulatory peptides, formed by the action of GK on some substrate(s) contained in the foetal calf serum present in our cultures. Under in vivo conditions, the proteolytic action of GK may result in decreased concentration of the same substrate(s) and in a deficient production of stimulatory peptides for a few days, until the substrates are reconstituted in full. Differences in GK action were also observed when the route of GK administration changed from subcutaneous to oral. In both situations, some suppression of immune reactions was observed if the treatment was simultaneous with or shortly preceded, antigen administration. On the other hand, the oral administration of GK appeared to enhance the induction of tolerance if given together with the oral antigen. The ensuing suppression of the arthritic reaction exceeded in duration and in magnitude the "immunosuppressive" effect of GK alone, administered either by injection or by mouth. The same experiments also demonstrated that oral tolerance could not be induced by antigen alone in sialoadenectomized animals. Oral tolerance is a state of antigen specific hyporesponsiveness which follows oral delivery of an antigen. It represents a protective reaction by the gut associated mucosal tissue (GALT) to prevent unnecessary and potentially harmful reactions to food antigens. It involves more than one mechanism. High antigen doses induce clonal deletion, while lower doses induce an active form of suppression or immune deviation, mediated by TGF- and IL-4 producing T cells,
234
6.5
6.0
E E v nLU i'LU
5.5
< a
.,,..=
n<<:: 5.0
4.5 4.3
..j/; 0 14 1'61'8 2'1'2'3;~5 28 31
,g :35:36
4'2 Days
DAYS AFTER IMMUNIZATION Figure 8. Effects of SMX and oral pGK on the induction of oral tolerance in Lewis rats. (0) immunized controls; (O) oral collagen followed by immunization; (A) SMX followed by immunization; (I) SMX, oral collagen, followed by immunization; (I-]) SMX, oral collagen, oral pGK, followed by immunization.
referred to by some authors as TH3 cells. The action of these cells suppresses TH1 responses and favors TH2 responses [17-19]. From Peyer's Patches, these regulatory cells migrate to periferal lymphoid organs rendering systemic the modified responsiveness. Thus, our results suggest that the SM gland plays a significant role in maintaining the normal responsiveness of GALT and that GK secretion in saliva is one of the factors involved in this function. The immunological properties of GK make this molecule an interesting candidate in the treatment of autoimmune diseases.
ACKNOWLEDGMENTS The experiments described here were carried out in collaboration with A. Kemp, L. Mellow, M. Abdelhaleem (experiments with crude SM gland extracts and with semi-purified rGK). Financial support was from the Medical Reasearch Council of Canada.
235
REFERENCES
.
.
.
.
.
.
10.
11. 12. 13. 14. 15. 16. 17.
18. 19.
Fuller PJ, Funder JW. The cellular physiology of glandular kallikrein. Kidney 1986; 29: 953. Wines DR, Brady JM, Pritchett DB, Roberts JL, MacDonald RJ. Organization and expression of the rat kallikrein gene family. J Biol Chem 1989; 264: 7653. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 1992; 44: 1. Alhenc-Gelas F, Marchetti J, Allegrini J, Corvol P, Menanrd J. Measurement of urinary kallikrein activity species differences in kinin production. Biochim Biophys. Acta 1981; 677: 477. Simpson JAV, Chao J, Chao L. Localization of kallikrein gene family proteases in rat tissues. Agents and Actions-Suppl 1992; 38 (Pt. 1): 595. Naughton MA, Geczy C, Bender V, Hoffman H, Hamilton E. Esteropeptidase and thymotropic activity of a protein isolated from the mouse submaxillary gland. B iochim Biophys Acta 1972; 263: 106. Hu ZQ, Murakami K, Ikigai H, Shimamura T. Enhancement of lymphocyte proliferation by mouse glandular kallikrein. Immunol Lett 1992; 32: 85. Perris AD, Whitfield JF. The mitogenic action of bradykinin on thymic lymphocytes and its dependence on calcium. Proc Soc Exp Biol Med 1969; 130: 1198. Ishizaka K. Twenty years with IgE: from the identification of IgE to regulatory factors for the IgE response. J Immunol 1985; 135: i-x. Verwaerde C, Auriault C, Neyrinck JL, Capron A. Properties of serine proteases of Schistosoma mansoni. Shistosomula involved in the regulation of IgE synthesis. Scand J Immunol 1988; 27: 17. Matsushita S, Katz DH. B iphasic effect of kallikrein on IgE and IgG1 syntheses by LPS/IL4 stimulated B cells. Cell Immunol 1993; 146: 210. Kemp A, Mellow L, Sabbadini E. Suppression and enhancement of in vitro lymphocyte reactivity by factors in rat submandibular gland extracts. Immunology 1985; 56: 261. Abdelhaleem M, Sabbadini E. Identification of immunosuppressive fractions from the rat submandibular salivary gland. Immunology 1992; 76: 331. Nagy E, Berczi I, Sabbadini E. Immunoregulatory effects of glandular kallikrein from the salivary submandibular gland of rats. Neuroimmunomodulation 1997; 4: 107. Edman P, Begg G. A protein sequenator. Eur J B iochem 1967; 1: 80. Trautschold I. Assay methods in the kinin system. In Erdos G: Handbook of experimental pharmacology, Bradikinin, kallidin, and kallikrein. Berlin, Springer, 1970; 25: 52-81. Weiner HL, Friedman A, Miller A, et al. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol 1994; 12: 809. Weiner HL. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997; 18: 335. Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci 1994; 91: 6688.