Using proteinase trapping to detect revertants of inactive rhinoviral 2A proteinase mutants.

Biol. Chem. Hoppe-Seyler Vol. 373, pp. 523-528, July 1992

USING PROTEINASE TRAPPING TO DETECT REVERTANTS OF INACTIVE RHINOVIRAL 2A PROTEINASE MUTANTS a MARION LuDERER , HANS-DIETER LiEBiGa, WOLFGANG SOMMERGRUBER^, DIETER BLAASS, ERNST KuECHLERa AND TIM SKERNa

Institute of Biochemistry, University of Vienna, Waehringerstrasse 17, A-1090 Vienna, Austria. ^Ernst Boehringer Institut fuer Arzneimittelforschung, Dr. Boehringergasse 5-11, A-1120 Vienna, Austria.

Summary The 2A proteinase of human rhinovirus 2 cleaves itself off the growing polyprotein at its own N terminus during translation; this property was used to develop an in vivo screening system with the lacZ gene fragment of M13mpl8. The fusion of an active 2A proteinase to the Cterminus of the α-fragment did not affect α-complementation, as the proteinase cleaved itself off the α-fragment. However, an inactive 2A proteinase remained fused to the α-fragment hindering α-complementation. Random mutations were then introduced into the 2A gene site by PCR amplification. Mutants defective in α-complementation (thus containing an inactive 2A proteinase) were obtained at an efficiency of 5%, mutants showing reduced 2A activity at an efficiency of 1%. Mutants showing reduced or no 2A activity were then subjected to PCR mutagenesis. Three mutants reactivating an inactive 2A proteinase were examined and the compensatory changes determined. Introduction The genetic information of human rhinoviruses (the main causative agents of the common cold[l]) and the closely related polioviruses is contained in a single large open reading frame. During translation, the polyprotein produced is processed cotranslationally; thus, the entire polyprotein is never observed [2]. The first proteolytic cleavage is carried out by the virally encoded proteinase 2A, which cleaves itself off its own N terminus [3,4]. All remaining cleavages are carried out by the second viral proteinase 3C, except that between the capsid proteins VP4 and VP2 [5]. This cleavage remains obscure [6]. The 2A proteinases of rhinoand polio-viruses are also involved in the shut off of host cell translation by catalyzing indirectly the cleavage of the p220 component of the eukaryotic initiation factor 4F [7,8]. From sequence comparisons, 2As have been classed as serine proteinases [9]; furthermore, the sequences can be modelled onto the three-dimensional structure of a-lytic Abbreviations; HRV, human rhinovirus; IPTG, isopropyl -D-thiogalactoside; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside; PCR, polymerase chain reaction.

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proteinase [10]. However, the active site nucleophile of the 142 amino acid long HRV2 2A proteinase is Cys:106; the other residues of the putative catalytic triad are His: 18 and Asp:35 [4]. In absence of crystallographic data, mutational analysis of the 2A should give insight into its structure and mechanism. The use of a genetic screening system [11] to detect revertants of inactive mutants is presented here. Materials and Methods Bacterial strains and plasmids The expression of the VP1-2A gene fragment of the HRV 2 cDNA was achieved using M13mpl8 as described [11). The first 45 amino acids of the lacZ gene (i.e. the α-fragment), 8 amino acids from the polylinker, 28 C-terminal amino acids of VP1 and all 142 amino acids of the rhinoviral 2A proteinase are expressed as a fusion protein. Propagation and expression of this construction was carried out in E.coli JM101 which carries a gene for -galactosidase lacking amino acids 11 to 41 (the M15 -galactosidase). Materials and General Methods Restriction enzymes and DNA modifying enzymes were from Boehringer Mannheim or New England Biolabs; Taq (Thermus aquaticus) DNA polymerase was from Promega. Manipulation and preparation of DNA was by standard protocols 112]. DNA sequencing was performed by using TV DNA polymerase from Pharmacia and [a-32p]dATP from Amersham. O.lomM IPTG as inducer and 0.8mM X-Gal as substrate (both from Boehringer Mannheim) were used for detection of α-complementation. Expression of proteins from M13 was carried out as described [12]. After induction at 37°C for 6 hrs the cells were harvested and the proteins were prepared for gel electrophoresis [11] on 15% polyacrylamide gels. The gels were blotted onto nitrocellulose (Schleicher & Schuell) and the blots probed with the antiserum PC20, directed against the last 20 C-terminal amino acids of the HRV2 2A proteinase [4] and with a second antibody coupled with alkaline phosphatase (Promega).

Results The basis of proteinase trapping A -galactosidase molecule lacking amino acids 11-41, referred to as M15 -galactosidase, can be complemented by a peptide containing this sequence to form an active enzyme [13]. This peptide (the α-fragment) comprises amino acids 3-92 of the lacL gene; it can, however, be further reduced in size without affecting α-complementation [14]. A genetical screening method based on this α-complementation system was then developed to allow the detection of a large number of proteinase mutants. The first 45 amino acids of the α-fragment of M13mpl8 were fused to a DNA fragment encoding the C-terminal 28 amino acids of VP1 and all 142 amino acids of an active or an inactive 2A proteinase, followed by two stop codons. The C-terminus of VP1 was included to ensure that 2A cleavage could take place. As an active 2A proteinase cleaves itself off its own


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N-terminus as soon as it is synthesized, the α-fragment should be freed and should undergo acomplementation. The fusion of an inactive 2A proteinase to the lacL gene fragment however leads to a C-terminal extension of the α-fragment; this hinders its diffusion and is therefore detrimental to α-complementation (Fig.l).

M13/2A+

M13/2A-

«i*»« B BH IslililisiPl E 45

28

142

α VP1 2A

45

28

142

α VP1 2A

α-complementation Figure 1. The logic of proteinase trapping. DNA fragments coding for an active (VP1, striped; 2A, circles) and an inactive 2A proteinase (indicated by the star) were fused to the afragment (hatched; the open region represents amino acids from the polylinker) of M13mpl8. An α-fragment freed by cis processing can undergo α-complementation with the M15 -galactosidase whereas one trapped by an inactive 2A cannot.

These two constructions containing genes coding for an active 2A proteinase or an inactive one (inactivation was by deletion of the glycine residues 104,107 and 108) were transformed into competent E.coli JM101 cells as described above. Plaques from the active 2A/M13 construction were dark blue whereas plaques from the inactive 2A construction were colourless. This restriction of α-complementation is referred to as proteinase trapping. Random PCR mutagenesis using Taqpofymerase and screening for mutations Random mutations were then introduced into the active VP1/2A DNA fragment by using the high error rate of the Taq DNA polymerase in the PCR reaction; the enzyme possesses a high error rate as it lacks a 3'->5'exonuclease proof-reading activity. Two primers starting outside the VP1-2A region were used in a standard PCR containing Ing DNA. After 30 cycles, a further 3 units of Taq DNA polymerase were added and a 10 more cycles performed. The resulting amplified DNA was then cleaved with restriction enzymes to give a 450-bp-fragment coding for the last 8 amino acids of VP1 and the entire 2A sequence; this randomly mutated fragment was then used to replace this sequence in the construction bearing the active proteinase. A library of phage containing mutated VP1-2A sequences was thus created. 5% of the plaques resulting from subsequent transformation of this DNA into E.coli JM101 cells were defective in α-complementation, as the plaques remained white. 1% of the plaques were


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faintly blue and the remainder were not distinguishable from wild-type plaques. Proteins were induced from each type of plaque with IPTG and were examined on Western blots. Proteins derived from white plaques all showed only the uncleaved form of 2A ; sequencing revealed that these mutants contained one to three mutations in the VP1/2A gene fragment. Proteins from three phages giving rise to faintly blue plaques also showed the 24kDa band of uncleaved 2A. It is assumed that the 2A proteinase is only marginally active; although insufficient mature 2A is produced to be detected on Western blot, a low level of acomplementation can still be detected. Amino acid changes in the 2A gene were found in all of these mutants. Proteins from phage giving rise to blue plaques all contained a band corresponding to mature 2A; the derived amino acid sequence of twelve such plaques was that of the wild-type. The remaining three had mutations which presumably do not affect function. The number and distribution of nucleotide changes were as described [11]; the amino acid changes of 6 2A genes bearing one single mutation are shown in Fig.2 together with 12 mutants from [11].

P2A

AVP1

(U2)

(28)

tr

here

gr

r wssds y

C YYCk

ss

c C C G G FFF

A A AAAA

-3

3

495259606162 83

His18 Asp35

AA A A AAA

ΙΟΙ 112 118123130 Cys106

Figure 2. Single amino acid changes detected in 18 2A genes. The HRV2 VP1/2A sequence is represented by the line; the position and nature of the changes in phage derived from blue plaques (solid), faintly blue plaques (crossed) or white plaques (open) are shown. Capital letters indicate residues conserved throughout all rhino- and entero-virus 2A sequences.


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Compensatory mutations can reactivate an inactive 24 proteinase Single-stranded DNAs from two white-plaque mutants and one light-blue plaque mutant at position 130 (PheiLeu, PheiSer and PherTyr respectively) were subjected to random mutagenesis to examine whether further mutations within the 2A could reactivate the enzyme. Blue plaques were found only with DNA from the mutant Phel30:Tyr; however, the plaques were not as intensely blue as those from DNA bearing a wild-type 2A. Analysis of proteins induced with IPTG by immunoblotting showed that mature 2A proteinase was present in extracts of the blue plaques, but not in extracts of the original mutant (Fig. 3A). DNA sequencing of 2A genes from several blue plaques showed that one of three mutations (Ser27:Pro, His 135:Arg or His 137:Arg; Fig. 3B) could compensate for the original mutation.

P2A

ΔΥΡ1 (28)

(142)

r r

P

ts

kD*

11

τ 27

γ t

tt h h

TT

111 135 137 130

Figure 3. Analysis of revertants from the mutant Phel30:Tyr. A: Western blot of proteins induced from JM101 cells transacted with the indicated M13 DNAs separated on a 15% polyacrylamide gel and probed with the PC20 antiserum. The open arrow indicates the 15 kD mature 2A and the closed arrow the 24 kD uncleaved product. B: Nature and position of the three compensating mutations found in the Phel30:Tyr revertants. Symbols are as in Fig.2.

Discussion The phenomenon of α-complemerrtation has been adapted to enable the high capacity screening of proteinase mutants. Use was made of the ability of picornaviral 2A proteinases to cleave themselves out of a growing polyprotein. In this case, the α-fragment is released and can undergo α-complementation without impairment. Any modification which inactivates the proteinase can trap the α-fragment by remaining fused to it and hindering its diffusion. This versatile system (which should be applicable to other proteinases which behave similarly) thus allows the screening of a large number of proteinase mutants.


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The random mutations inactivating HRV2 2A were found mostly at residues conserved in all picornaviruses. Furthermore, a significant fraction was clustered at the C-terminus of the enzyme, with the amino acid Phel30 being especially sensitive. Even the presence of TyrlSO was sufficient to inactivate almost completely the enzyme. The role of this region in 2Acatalysed proteolysis is however not clear, as there is no similarity to serine proteinases [11]. Further evidence for the importance of this domain came from revertants which relieved the block caused by the mutation Phel30:Tyr; two of the compensating mutations (His 135:Arg and His 137: Arg) lay very close to the original mutation. Revertants from phage bearing the changes Phel30:Ser and Phel30:Leu could not be found under the conditions used, suggesting that the structural changes wrought by these mutations are too severe to be overcome by a single mutation. The detection of the revertants demonstrates the power of proteinase trapping. By examining further revertants at this and other positions, it should be possible to build a map of interactions within the enzyme and thus to test whether proposed structures of this class of enzymes are valid [11]. Acknowledgements We thank G. Casari for modelling studies. This work was supported by the "Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung" and by Boehringer Ingelheim.

References l.Stott,E.J. & Killington. R.A. (1972^ Ann. Rev. Microbiol. 26. SQ3-524. 2. Hellen, C.U.T., Kräusslich, H.-G. & Wimmer, E. (1989^ Biochemistry 28. 9881-9890. 3. Toyoda, H., Nickiin, M.J.H., Murray, M.G., Anderson, C.W., Dunn, J.J., Studier, F.W & Wimmer, E. (1986) Gell 45, 761-770. 4. Sommergruber, W., Zorn, M., Blaas, D., Fessl, F., Volmann, P., Maurer-Fogy, L, Pallai, P., Merluzzi, V., Matteo, M., Skern, T. & Kuechler, E. (1989 Virology 169, 68-77. 5. Hanecak, R., Semler, B., Anderson, C.W., & Wimmer, E. (1982) Proc. Natl. Acad. Sei. USA 79,3973-3977. 6. Arnold, E., Luo, M., Vriend, G., Rossmann, M.G., Palmenberg, A.C., Parks, G.D., Nickiin, M.J.H. & Wimmer, E. 987^ Proc. Natl. Acad. Sei. USA 84. 21-25. 7. Etchison, D., Hansen, E., Ehrenfeld, E., Edery, L, Sonenberg, N., Milburn, S., & Hershey, J.W.B. (1984) J.Virol. 51, 832-837. 8. Kräusslich, H.-G., Nickiin, M.J.H., Toyoda, H., Etchison. D. & Wimmer, E. (1987) J.Virol. 61,2711-2718. 9. Gorbalenya, A.E., Blinov. V.M. & Donchenko, A.M. (1986) FEBS Lett. 194, 253-257. 10.. Bazan, F. & Fletterick, R. (1988) Proc. Natl. Acad. Sei. USA 85, 7872-7876. 11. Liebig, -D., Skern, T., Luderer, M., Sommergruber, W., Blaas, D. & Kuechler, E. (1991) Proc. Natl. Acad. Sei. USA 88,5979-5983. 12. Sambrook, J., Fritsch, E. & Maniatis, T. (1987) Molecular Cloning: a Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor. 13. Langley, K.F., Villarcjo, M.R. Fowler, A.V., Zamenhof, P.J., & Zabin, I. (1975) Proc. Natl. Acad. Sei. USA 72, 2154-2157. 14. Vieira, J. & Messing, J.G. (1982) Gene 19,259-268.


Biol. Chem. Hoppe-Seyler Vol. 373. pp. 529-546. July 1992

Migration of Polymorphonuclear Leukocytes through Human Amnion Membrane · A Scanning Electron Microscopic Study BIRGIT BAKOWSKI AND HARALD TSCHESCHE

University of Bielefeld, Faculty of Chemistry, Department of Biochemistry, Universitätsstraße, P.O. Box 8640, D-4800 Bielefeld 1, F.R.G.

Summary Scanning electron microscopy has been used to examine the changes in the basement membrane structure induced by polymorphonuclear leukocytes during leukodiapedesis. A diapedesis model based on a Boyden chamber fitted with a human amnion membrane simulated inflammatory processes during which leukocytes are stimulated to leave blood vessels and to penetrate basement membranes. In the Boyden chamber, a chemotactic gradient was developed from a solution of 10"^ M formylmethionyl-leucyl-phenylalanine, which induced the cells to migrate through the membrane. Our observations suggest that leukocytes, like tumor cells, emigrate in a three-step process. In the first instance, they adhere to the basement membrane. A local partial proteolysis follows, which is caused by secreted metalloproteinases, especially by gelatinase. The partial dissolution of the matrix facilitates cell penetration. Active locomotion and squeezing through the residual membrane matrix allows the cells to penetrate without complete local membrane destruction. The last step involves migration of the cells through the connective tissue barrier to the site of infection. The type I collagen fibres which form this loose stroma tissue are no obstacle and can be pushed aside without proteolytic degradation.

Introduction Infectious microorganisms can invade a host and cause inflammatory processes. The host starts defence reactions whose functions are to stop and eliminate these foreign organisms and to repair the damaged tissue [9]. Keywords: Polymorphonuclear leukocytes - basement membranes - leukodiapedesis - inflammation - scanning electron microscopy

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Inflammation is characterised by an accumulation of polymorphonuclear leukocytes (PMNL) at the site of injury. Physiologically, they are the most important effector cells in the defence system against invading microorganisms. For this purpose, PMNL have the ability to leave the peripheral circulation in response to chemotactic stimuli generated by bacterial products, such as formyl-peptides, cell debris or damaged tissue (leukodiapedesis) [24]. The first phenomenon observed in leukodiapedesis is an increased adherence of PMNL to endothelial cells which is mediated by a leukocyte membrane glycoprotein, the Cdw 18-complex [12]. After migration through intercellular junctions [6], PMNL penetrate the basement membrane and migrate through the connective tissue along a chemotactic gradient to the site of infection (chemotaxis) [39,43]. After interaction of chemotactic agents with specific cell receptors, PMNL are induced to change their morphology by reorganization of their microfilament system [34] leading to a visible polarization and a ruffled surface of the cells. The migration of PMNL is accompanied by exocytosis of specific granules [42]. The content of these granules, e.g. type I collagenase and/or gelatinase, is capable of degrading extracellular matrix components such as type IV collagen, fibronectin and others. [1,8,14,19,20,25,33,38]. Arriving at the site of injury PMNL immediately start to eliminate opsonized particles by ingestion and internalization into a phagosome. Granules, present in the cytoplasma of PMNL [41], are transported by the microtubule system [26] and fuse with the phagosome. In the phagolysosome thus formed foreign material can be digested by the granule enzymes (phagocytosis) [17]. The massive non-physiological release of granule enzymes can result in pathophysiological processes, such as rheumatoid arthritis [40]. The details of the mechanisms involved in leukodiapedesis, especially the penetration of PMNL through the basement membrane, are still unknown. Russo et al. [27] describe a useful model based on a modified Boyden chamber in which the two compartments are separated by a micropore filter with an overlaying amnion membrane serving as a basement membrane with epithelial cell layer and underlying stroma tissue. When the lower compartment is filled with a solution of formyl-peptides, migration of PMNL through the membrane is induced. This in vitro system enabled us to study the extravasation pathway of PMNL by scanning electron microscopy and allowed us to obtain a three-dimensional impression of PMNL during penetration of a basement membrane.


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Material and Methods Isolation ofPMNL Blood was taken by venipuncture from healthy volunteers. PMNL were isolated from the heparinized blood in two steps according to a method of Boyum [2]: erythrocytes were separated by sedimentation with the Ultravist-Methocel-method (18.65 % Ultravist, Schering, Berlin; 1.45 % Methocel, Sigma, M nchen, in H20, bidest.). A density gradient centrifugation with Histopaque (Sigma, M nchen) resulted in a granulocyte-rich pellet with approximately 99 % intact and living PMNL as determined from trypan dye exclusion. Prior to use PMNL were suspended in DPBS (-) /pH 7.3 (0.14 M NaCl, 0.0027 M KC1, 0.008 M Na2HPO4-2H2O, 0.0015 M K2PO4, 0.5 mM MgCl2-6H2O, 0.9 mM CaCl2'2H20,0.1 % glucose). No lysis buffer was used. Preparation of human amnion membranes Frozen postnatal amnion membranes were obtained from a local hospital. After thawing the membranes were washed with CMF-PBS/pH 7.3 (0.14 M NaCl, 0.0027 M KC1, 0.008 M Na2HP04-2H2O, 0.0015 M KH2PO4). Their three-layered structure is depicted in Fig. 1. To observe the surface of the basement membrane, the epithelial cell layer was lysed with 4 % deoxycholate solution (Sigma, M nchen) and removed mechanically, according to a method of LlOTTAetal. [22]. Modified Boy den chamber Fig. 2 shows, schematically, the two-compartment chemotaxis chamber described by Russo et al. [27] but made of Teflon by the mechanical workshop of the University of Bielefeld. The upper compartment was filled with 200 μΐ PMNL suspension, the lower with ΙΟ"7 Μ FMLP (formylmethionyl-leucyl-phenylalanine, Sigma, M nchen) in DPBS (-). The two parts were separated by a micropore filter (pore size 5 μιη, Millipore, Molsheim) superimposed by a human amnion membrane. The chambers were placed in sterile petri dishes. After incubating the amnion membrane with PMNL at 37°C for 3 h it was prepared for SEM. Inhibition assay Boyden chambers were prepared in the manner described above. However, the amnion membrane was preincubated with 0.1 μg TIMP (tissue inhibitor of metalloproteinases, a gift from Dr. G. MURPHY, Cambridge, England) for 15 min. in five-fold excess before cells were seeded into the upper compartment. The amount of TIMP corresponded to the total content of gelatinase of 10^ cells (personal communication A. SCHETTLER). Brought to you by | Purdue University Libraries Authenticated Download Date | 5/25/15 7:53 PM

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Three-layered structure of a human amnion membrane

E

Epithelial cell layer

B

Basement membrane

S

Stroma tissue

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sy*

/Λ^-*^\j^^S~^>^^^~^-^~\^~^ —^— ν,^χ—s— '-V-/-N^

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A M N I O N M E M B R A N EE ^ -

—

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\ ^}·

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Fig. 2. Amnion chemotaxis chamber, description see text

Scanning Electron Microscopy Fixation was performed at 4° C with 3 % glutaraldehyde (Serva, Heidelberg) in 0.1 M cacodylate buffer/pH 7.3 for 2 h. The specimens were postfixed with 0.5 % OsC>4 (Serva, Heidelberg) in 0.1 Μ cacodylate buffer/pH 7.3 at 4°C for 2 h and dehydrated through a graded series of ethanols. After critical point drying the specimens were sputter-coated with gold (layer thickness 400 A). Scanning electron micrographs were taken on the Hitachi S-450 apparatus using Agfapan 100 professional films. The apparatus was operated at 15 kV. The specimens were viewed at 45° beam incidence.

Results and Discussion Scanning electron microscopic investigations on polymorphonuclear leukocytes penetrating basement membrane were performed on human amnion membrane fitted to a modified Boyden chamber. The lower compartment of the Boyden chamber was filled with a 10"7 M solution of FMLP and the upper part with a suspension of PMNL. Thus, the amnion membranes were exposed to the cells migrating towards the developing FMLP gradient. The amnion membranes were investigated by electron microscopy three hours after the experiment was started. This system described by RUSSO et al. [27] in combination with scanning electron microscopy allowed the attainment of a three-dimensional impression of the interaction of PMNL with the natural matrix barrier of a basement membrane. The amnion membrane with its three-layered structure, Brought to you by | Purdue University Libraries Authenticated Download Date | 5/25/15 7:53 PM

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epithelial cell layer, basement membrane and loose stroma tissue (Fig. 1), serves to mimic the traversal pathway of PMNL leaving blood vessels, especially the penetration of basement membranes. An unstimulated cell shape is characterised by a spherical form with a smooth surface (not shown). Cells stimulated by an FMLP gradient developed through the amnion membrane change their morphology, whereas PMNL express a great variety of cell shapes. Figure 3 shows a typical example with a knoblike tail and lamellipodia in front. In contrast to the ruffled surface of the front region the tail exhibited a smooth exterior. The arrow on Figure 3 indicates a lamellipodium between the dense network of the basement membrane. Although this is a stationary picture it leads us to the impression of a PMNL pushing a lamellipodium between the matrix. After interaction with chemotactic agents PMNL are stimulated accompanied by morphological changes which are well documented by our scanning electron micrographs. The rapid polarization of the cells within a few seconds [18,34] is accompanied with enlargement of the cell surface. Therefore, it can be assumed that the required cell membrane material is derived from intracellular membranes. HOFFSTEIN et al. [18] demonstrated the association between surface enlargement, endocytic activity and loss of specific granules in the cytosol of PMNL. The expression of ruffles in the front region of the cells can be explained by the same mechanism. Fusion of storage granules with the outer cell membrane after activation increases membrane material leading to ruffles. Neutrophil activation via FMLP receptors and the processes involved are well characterised [30]. Recently, SINGER et al. [29] identified four types of extracellular matrix receptors for laminin, C3 bi/fibrinogen, fibronectin and vitronectin which are located on the inner surface of the specific granule membrane. After activation with e.g. FMLP, these specific granules ("adhesomes") fuse with the cell membrane. This leads in a regulated event to expression of the extracellular matrix receptors on the surfaces of PMNL and allows cell attachment during inflammatory processes. Figure 3 is the first in a series of pictures which demonstrate the steps involved in traversing of PMNL through basement membrane. A similar process must be expected for the extravasation of tumour cells during metastasis, as based on a hypothesis of LIOTTA et al. [21,23,32] who postulated a three-step mechanism: 1. Adhesion to the basement membrane, 2. local dissolution of the membrane, and 3. locomotion through the membrane and in the connective tissue. Indeed, the initial event observed is the adherence of PMNL which obviously prefer hollows in the basement membrane for settlement, as seen in Figure 4. Our scanning electron microscopic observations suggest two possibilities for creating hollows in the membrane. Either PMNL use natural grooves or they actively change the morphology of the basement membrane. In addition, PMNL often accumulate at tissue already damaged before migrating through this natural barrier (Fig. 4). Brought to you by | Purdue University Libraries Authenticated Download Date | 5/25/15 7:53 PM

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Polarized PMNL on human amnion membrane

The arrow indicates a lamellipodium between the dense network of the basement membrane,

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Fig. 4.

Accumulation of PMNL at tissue already damaged

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The following pictures demonstrate the second step of penetration: PMNL partially degrade the dense network of the type IV scaffold of the membrane. A polarized cell with ruffled surface which seems to begin lysing the matrix fibres is seen in Figure 5. The next step is depicted in Figure 6: A partial degradation of fibres closely surrounding the cell can be observed. The damage to the membrane is limited to the immediate environment of the cell, also observed during interactions of tumour cells with the basement membrane [21,23,32]. The local destruction leads to loss in fibre density, as shown in Figure 7, and enables the cells to slip into the tissue. Local partial lysis of the basement membrane barrier obviously facilitates penetration. WRIGHT and GALLIN [42] demonstrated that, during migration, PMNL release the content of specific granules by exocytosis in response to chemotactic stimuli. These findings are confirmed by our investigations of the FMLP-stimulated release of collagenase and gelatinase [34], whereas gelatinase is the first enzyme to be secreted and detected in the extracellular environment of PMNL. A sequential secretion of collagenolytical enzymes can be assumed [15], which enables cells to regulate the degradation of the extracellular matrix. VISSERS and WINTERBOURN [36] showed that the degradation of basement membranes is caused by gelatinase. We observed an increased amount of gelatinase in the supernatant of FMLP-stimulated PMNL in the Boyden chamber (data not shown) and can support the in vitro investigations of VISSERS et al. [37] and UlTTO et al. [35] of gelatinolytical degradation of basement membranes. These results and scanning electron microscopic observations lead us to the conclusion that PMNL are able to enzymatically lyse basement membranes. Involvement of metalloproteinases, especially gelatinase, was further supported by inhibition experiments. The amnion membranes were preincubated for 15 min. with a five-fold excess of TIMP with regard to the total content of gelatinase. No local lysis of tissue fibres could be observed. The cell, Figure 8, seems to be unable to enter into the membrane. Investigations of the reverse sides of the amnion membrane showed that only a few cells crossed the membrane. Quantitative studies are under way in our laboratory. Therefore, proteolytic degradation is generally controlled by proteinase inhibitors. The most important inhibitor in this case is TIMP-1 [4,7,10,13] and/or TIMP-2 [11,31] (tissue inhibitor of metalloproteinases). Thus, tissue destruction will be limited to a region close to the cell environment, which is well documented by our investigations using scanning electron micrography. An excess of TIMP leads to an inhibition of cell migration. This provides strong evidence for the participation of metalloproteinases in the process of leukodiapedesis. In vivo, penetration of basement membranes must proceed in the presence of proteinase inhibitors, which are produced by a variety of cells [4,7,10,13]. CAMPBELL and CAMPBELL [5] proposed a mechanism by which lysis of matrix is limited to the pericellular space between the inflammatory


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Polarized PMNL, starting to lyse tissue

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Damage of tissue is limited to the immediate environment of the cell

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Loss of fibre density

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cell and the matrix. Thus, cells are able to degrade matrix components in the presence of proteinase inhibitors by locally high concentrations of proteinases in their micro-environment. Though an enzymatic proteolysis of basement membrane components seems to be a prerequisite for migration, it cannot be excluded that PMNL use mechanical forces for penetration. Involvement of both local enzymatical degradation (weakening of the type IV collagen network) and active mechanical dilatation of the fibre matrix by a moving cell, as indicated in Figure 9, shows a momentary picture of a PMNL locomoting through the membrane. After crossing the basement membrane barrier PMNL appeared in the loose meshwork of the stroma tissue (Fig. 10), which did not seem to be an obstacle in the further process of emigration of PMNL through the loose stroma tissue to the site of infection. Our observations indicated that little or no destruction of type I collagen fibres of stroma tissue took place. These findings are in agreement with the results of BROWN [3] and SCHMALSTIEG et al. [28], who proposed two mechanisms by which the locomotion of PMNL can be explained. The two-dimensional locomotion is adherence-dependent. This mechanism can be suitable for the process of emigration from blood vessels and penetration of basement membranes. Motility in a three-dimensional collagen gel is adherence-independent, which is adequate for locomotion in the extracellular stroma tissue of type I collagen fibres. The invasion of tissue takes place without proteolytic degradation of collagen fibres [3], which is in accordance with our observations. Further studies are required to show how regulation and activation of the proteolytic enzymes, which are secreted in a latent form [16], are achieved in vivo.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, special research programme SFB 223. The authors wish to thank Mrs. G. DELANY for linguistic advice.


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Fig. 9.


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Involvement of enzymatic degradation and active mechanical dilatation of the fibre matrix

bar: 5 μπι


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B. Bakowski and H.Tschcsche

Fig. 10.

After crossing the basement membrane PMNL invade the stroma tissue

bar:

5 μτη

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Proteinase trapping: screening for viral proteinase mutants by alpha complementation.

Proteinase mutants of Saccharomyces cerevisiae.

Determinants of substrate recognition by poliovirus 2A proteinase.

A method to detect proteinase activity using unprocessed X-ray films.

Proteinase inhibitors.

Proteinase-3 (PR-3): a polymorphonuclear leukocyte serine proteinase.

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Proteinase inhibitors from potatoes.

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The human multicatalytic proteinase: affinity purification using a monoclonal antibody.

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Proteinase inhibitors from plant sources.

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Proteinase inhibitors from legume seeds.

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Proteinase inhibitors from cereal grains.

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