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Targeting kallikrein 6 proteolysis attenuates CNS inflammatory disease

SACHIKO I. BLABER, BOGOLJUB CIRIC^*, GEORGE P. CHRISTOPHI, MATTHEW J. BERNETT, MICHAEL BLABER, MOSES RODRIGUEZ^*,, and ISOBEL A SCARISBRICK^,,,2

Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, USA;
Program for Molecular Neuroscience and Departments of
^* Immunology,
Neurology, and
Physical Medicine and Rehabilitation, Mayo Medical and Graduate Schools, Rochester, Minnesota, USA

²Correspondence: Department of Neurology, 428 Guggenheim Bldg., Mayo Clinic, 200 First St., S.W., Rochester MN 55905, USA. E-mail: Scarisbrick.Isobel{at}Mayo.edu

SPECIFIC AIMS

The primary goal of this study was to test the hypothesis that a newly identified serine protease, kallikrein 6 (K6), participates in CNS inflammatory disease. We evaluated whether autonomously generated K6 antibodies alter the clinicopathological course of disease in murine proteolipid protein139-151-induced experimental autoimmune encephalomyelitis (PLP139-151 EAE).

PRINCIPAL FINDINGS

1. K6 expression is up-regulated in activated immune cells
The hypothesis that K6 proteolytic cascades participate in inflammatory CNS demyelination stems partly from its abundant expression by inflammatory cell subsets within CNS perivascular cuffs and at sites of CNS demyelination in animal models of multiple sclerosis (MS) and in the human disease. However, whether lesional K6 expression represents constitutive basal levels or a frank up-regulation had not been addressed. We examined this by determining relative levels of K6 mRNA in resting and activated splenocytes. We have shown that activators of immune cell function such as concanavalin A or CD3 receptor cross-linking produces a >2-fold increase in K6 mRNA expression. These findings indicate that activated T cells such as those seen in CNS inflammatory lesions express higher levels of K6 than resting cells, which we propose contributes to pathogenesis.

2. Immunization of mice with recombinant K6 results in the generation of K6 function blocking antibodies capable of blocking K6 enzymatic activity in vitro
To determine the ability of antibodies generated by K6 immunization to block K6 enzymatic activity, we isolated immunoglobulin from pooled sera of mice immunized with K6 in complete Freund’s adjuvant (CFA), or with CFA alone, collected before EAE induction and determined its ability to inhibit K6-mediated hydrolysis in vitro. The IgG fraction from K6 immunized mice blocked K6-mediated degradation of myelin basic protein (MBP), reduced the rate of K6-mediated AcATRpNA substrate hydrolysis (Fig. 1 a, b), and inhibited invasion of activated splenocytes in a modified Boyden chamber assay (Fig. 1c ). Cumulatively, these experiments demonstrate that K6 immunization results in the production of antibodies able to block K6-mediated enzymatic activity, including that which may be responsible in vivo for hydrolysis of myelin proteins and facilitation of immune cell extravasation.

View larger version (27K): [in this window] [in a new window]   Figure 1. Immunoglobulin isolated from K6 immunized mice blocks K6 enzymatic activity and immune cell invasion in vitro. a) 20% Tricine SDS-PAGE showing hydrolysis of rat MBP in the presence or absence of IgG isolated from K6 immunized mice or controls. Lanes: 1, molecular mass markers; 2, K6 IgG + K6 + MBP; 3, CFA IgG + K6 + MBP; 4, K6 + MBP; 5, K6 IgG + MBP; 6, CFA IgG + MBP; 7, MBP alone (2 major bands shown at arrowheads). Incubation of MBP with K6 for 3 h resulted in hydrolysis (lane 4, see fragment at arrow). Preincubation of K6 with IgG isolated from K6 immunized mice blocked K6-mediated hydrolysis of MBP (lane 2) whereas IgG isolated from animals immunized with CFA alone (lane 3), did not. b) Rate of AcATRpNA substrate hydrolysis over time by K6, in the presence of IgG isolated from K6 immunized mice or controls. The presence of K6 IgG reduced the rate of substrate hydrolysis by 92.3% to a level similar to that seen in the absence of any added K6. c) Activated splenocytes were labeled with calcein AM and cultured in the upper well of a Boyden chamber with or without the addition of control or K6 IgG. Measurements of fluorescence in the lower chamber after 24 h indicated that addition of K6-IgG inhibited invasion by 20% vs. the addition of normal mouse IgG (m-IgG) or no antibody (none) (*unpaired Student’s t test, P0.005). Data represent mean ± SE of triplicate wells from an individual experiment; similar results were obtained from 3 independent experiments.

3. The presence of K6 function blocking antibodies was correlated with attenuation of both clinical and histological signs of EAE in PLP139-151-primed SJL/J mice
The goal of these experiments was to evaluate whether K6 immunization and generation of K6 function blocking antibodies could attenuate disease in an immune-mediated model system that parallels in many respects human MS. Our results indicate that immunization of mice before or at the time of PLP139-151 EAE induction significantly attenuated development of clinical and histological signs of disease. The mean day of disease onset, mean time to peak disease, mean daily clinical score after the onset of clinical signs, and mean maximal clinical disease score were all significantly reduced in K6 immunized mice relative to controls (Mann-Whitney U-test, P<0.04). The presence of high K6 antibody titers was associated with an increase in the number of days free of clinical signs (unpaired Students t test, P<0.001) and in a high-dose PLP139-151 EAE paradigm, with a significant increase in survival to the 21 day endpoint (X², P=0.003, Fig. 2 ). We have additionally demonstrated in a second model of EAE induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) that administration of a cocktail of two K6-specific monoclonal antibodies similarly attenuates clinical disease.

View larger version (16K): [in this window] [in a new window]   Figure 2. Immunization with K6 at the time of PLP139-151 EAE induction delays the onset and attenuates the severity of disease. Mice were immunized with PBS or K6 at the time of priming with 100 µg of PLP139-151 in CFA. a) K6 immunization at the time of priming was associated with a significant increase in survival (22/28 compared with 10/28, P=0.003 X2 using Fisher’s exact test). b) Data represent mean clinical score (±SE) of each group plotted against time. Clinical scores of mice immunized with K6 were significantly below those of PBS immunized mice from D9 throughout the remainder of the disease course examined (*P<0.01, Mann-Whitney U test).

Analysis of histological disease, as measured by quantitative evaluation of the percent of spinal cord quadrants with meningeal inflammation or parenchymal pathology at 12 or 21 days post-EAE induction, revealed significant reductions in each parameter in K6 immunized mice compared to controls (unpaired Student’s t test, P0.05). These results indicate that K6 immunization and the K6-specific antibodies generated delayed and attenuated clinical and histological signs of EAE and support the hypothesis that K6 is a mediator of CNS inflammatory disease.

4. The presence of K6 function blocking antibodies was correlated with reduced Th1 responses in vivo and in vitro
We examined effects of K6 immunization on the development of T helper 1 (Th1) responses. In vivo, PLP139-151-induced ear swelling (delayed-type hypersensitivity response, DTH) was suppressed in K6 immunized mice by 2.6-fold relative to immunization controls (unpaired Students t test, P0.003). To examine the potential effects of K6 immunization on Th1 cell expansion and/or differentiation, we assessed the level of PLP139-151-specific T cell proliferation and cytokine production in cultures of splenocytes isolated from K6 immunized mice. The proliferative response and production of IFN- in response to secondary stimulation with the priming antigen were significantly decreased in splenocytes isolated from mice immunized with K6. Greater than 10-fold more PLP peptide was required to produce equivalent proliferation by splenocytes isolated from K6 immunized mice compared to controls (unpaired Students t test, P0.005). Secretion of the Th1 cytokine IFN- was decreased by 3-fold in the K6 preimmunized mice (unpaired Students t test, P<0.05); IL-2 was unchanged. These results are consistent with the idea that K6 immunization reduces Th1 responses.

CONCLUSIONS

This study provides the first direct in vivo and in vitro evidence supporting the hypothesis that K6 is a mediator of CNS inflammatory disease. K6 is a "trypsin-like" secreted serine protease cloned in our laboratory that we and others have shown to be abundantly and preferentially expressed by neurons and oligodendroglia of the normal adult brain. In CNS inflammation however, we have observed abundant K6 expression by perivascular and infiltrating inflammatory cells. These observations have given rise to the hypothesis that while K6 plays roles in CNS homeostasis; in the case of inflammation, an excess introduced by infiltrating immune cells sets up an environment where widespread hydrolysis of CNS proteins, including myelin, can ensue. We provide evidence that K6 immunization and the generation of function-blocking K6 antibodies during the development of CNS inflammatory disease modulate the peripheral Th1 immune response, suppress immune cell trafficking, and down-regulate inflammatory processes in the peripheral immune compartment and CNS parenchyma (Fig. 3 ). These findings support the idea that K6 is a pleiotropic enzyme and that inhibiting its activity modulates CNS inflammatory disease at several levels of the inflammatory axis, resulting in disease attenuation.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram of the potential sites of action of K6 in the development of clinical and histological EAE. Data support the hypothesis that K6 immunization results in generation of K6-specific antibodies that block K6-mediated enzymatic activity at one or more levels of the neuroinflammatory axis. Our results suggest that K6 proteolytic cascades may mediate immune cell activation, Th1 differentiation, and/or immune cell trafficking within the periphery, hydrolysis of ECM components at the level of the BBB, and/or hydrolysis of proteins within the parenchyma of the CNS. These sites of K6 action are not mutually exclusive; K6 proteolysis or attenuation of K6 activity would have feed-forward and/or feedback effects at other levels (arrows).

The most significant finding of these studies was that K6 immunization resulted in attenuation of clinical and histological signs of EAE in PLP139-151-primed mice. In K6 immunized mice receiving high-dose PLP139-151, we also observed a significant increase in survival. The reduced mortality associated with the presence of K6 function-blocking antibodies underscores the potential beneficial effects of K6 inhibition in reducing the progression of disease in mice with PLP139-151-induced EAE. The K6 immunization strategy did not, however, prevent the eventual development of disease: clinical scores in the different groups were similar near the 21 day endpoint. It will be important to establish whether K6 inhibition after disease onset similarly influences clinical severity, whether more complete K6 inhibition would further inhibit disease progression, and if K6 inhibition combined with other therapeutics would have an additive effect.

While the mechanism by which K6 immunization results in attenuated disease has not been firmly established, we present evidence that this is likely to occur at multiple levels of the neuroimmune axis and to be mediated by the establishment of circulating anti-K6 antibodies that are function blocking. In previous studies we have demonstrated that substrates relevant to the pathogenesis of CNS inflammatory disease are readily cleaved by K6. We show here that IgG isolated from K6 immunized mice blocks the ability of K6 to degrade complex proteins such as MBP as well as small molecule substrates in vitro. We also show that IgG from K6 immunized mice diminishes inflammatory cell invasion in vitro. It follows that, in vivo, K6 antibodies would be able to inhibit K6-mediated immune cell extravasation into the CNS and K6-mediated tissue destruction within the brain parenchyma.

The function of elevated K6 in activated immune cells is not fully understood. As a secreted serine protease capable of hydrolyzing extracellular matrix (ECM) proteins, it is likely to participate in immune cell trafficking, including immune cell extravasation. Results presented here suggest that immune cell K6 may have additional roles in immune cell function, including activation, expansion, and/or differentiation of Th1 cells specific for the disease-initiating peptide.

The data presented support the hypothesis that K6 is a key participant in the development of CNS inflammatory disease and suggest several possible convergent proteolytic cascades by which this occurs, including K6-mediated activities in the priming and effector phases of disease pathogenesis. The present studies firmly place K6 alongside other serine proteases and matrix metalloproteases as a key effector molecule mediating neuroinflammatory demyelinating disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1212fje;

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This Article Full Text (PDF) All Versions of this Article: 18/7/920 03-1212fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by BLABER, S. I. Articles by SCARISBRICK, I. A Search for Related Content PubMed PubMed Citation Articles by BLABER, S. I. Articles by SCARISBRICK, I. A