Elizabeth J. Roemer

Interaction of Matrix Metalloproteinases with Serine Protease Inhibitors

Proteolytic activity is responsible for inflammatory damage to connective tissues in a number of acute and chronic processes involving such diverse organs as the lungs, heart, joints, skin, and periodontiurn. Regardless of the nature of the inflammatory stimulus, a significant contribution to the tissue damage associated with inflammatory injury appears to come from unregulated degradation by the proteases from activated leukocytes. Neutrophil elastase (NE) is an especially potent protease which can degrade multiple components of the interstitial matrix in vitro and which has been implicated in inflammatory tissue damage in vivo. In the plasma and the interstitial stroma, the major endogenous defense against excessive NE activity comes from al-antitrypsin (aI-protease inhibitor, q-PI), a plasma serine protease inhibitor, or serpin, which is present in interstitial fluid at about half its normal plasma concentration of 1-2 mg/mL. We recently demonstrated that al-PI can be bound tightly to a complete interstitial extracellular matrix (ECM) in vitro with retention of its antielastase activity.’ Such ECM-bound antiprotease activity could contribute, along with fluid phase inhibitor, to the overall defenses against proteolytic damage to stromal tissues in vivo. Several laboratories have shown that fluid phase aI-PI can be completely inactivated by proteolytic cleavage of a constrained loop in the native molecule. A number of matrix metalloproteinases (MMPs), such as neutrophil collagenase (MMP-8), can proteolyze the loop in fluid phase al-PI at specific sites” in addition to cleaving matrix proteins. In this communication, we have extended the earlier fluid phase studies to ECM-bound aI-PI to determine whether matrix binding affects the sensitivity of this serpin to inactivation by MMPs. It is our hypothesis that inhibition of the MMP-8 activity of activated neutrophils could be of therapeutic value in helping to sustain the normal protective serpin levels within the interstitium while also blocking collagenolytic activity. One such class of inhibitors of MMP activity that could be useful for this application is the tetracy-

Cholinergic modulation of immunoglobulin secretion from avian plasma cells: the role of calcium.

The existence of a functional connection between the nervous and immune systems has long been argued. To determine if such a link exists in the secretory immune system, we have examined the avian lacrimal gland (Harderian gland) which contains large numbers of plasma cells. We have shown that these plasma cells bind an antibody to muscarinic acetylcholine receptor and that carbachol, an acetylcholine agonist, increases the secretion rate of IgG by these cells above a constitutive baseline level. This neurotransmitter-dependent increase of immunoglobulin secretion requires an influx of Ca2+, whereas the constitutive baseline secretion is apparently less dependent on such a flux. Furthermore, the Ca2+ flux appears to be mediated by voltage-dependent calcium channels. These data support the hypothesis that plasma cells can respond to neurotransmitters and, in the case of acetylcholine, increase immunoglobulin secretion.

Protective antigen composite nanofibers as a transdermal anthrax vaccine

Anthrax, a disease caused by the gram positive bacteria Bacillus anthracis, has become an increasing threat to public health in the last several years, due to its use as an agent of biological warfare. The currently utilized human anthrax vaccine, which confers immunity through the host antibody recognition of protective antigen (PA), requires a three dose regimen and annual booster shots after the initial vaccination to maintain its efficacy. The long term goal of this project is to produce an anthrax vaccine that is capable of delivering protective antigen through human skin. The novel method for transdermal vaccine delivery that we propose utilizes the high surface area to volume ratio offered by protein-containing nanofiber membranes, prepared by the electrospinning technique. Research has already been undertaken to study the effect the main virulent agent of anthrax, lethal toxin (LT), has on a human monocytic cell line, Monomac 6 cells (MM6). Lethal toxin is said to comprise of a Zn2+-dependent metalloprotease known as lethal factor (LF), and a binding protein known as protective antigen. The successful encapsulation of the protective antigen within the nanofibrous membrane was analyzed with the use of an in vitro MM6 assay. The assay was designed to ensure the functionality of PA through the harsh environment of the electrospinning process. Quantitative analysis of IL-6 cytokine production by lipopolysaccharide (LPS) stimulated MM6 cells in the presence of LF and PA provided proof that PA retained its biological activity through the process of electrospinning. This finding provides an innovative platform for the development of a transdermal anthrax vaccine.

Maxi-K channels in plasma cells

Whole-cells, excised outside-in and outside-out membrane patches were employed to study the electrophysiological properties of plasma cells isolated from the Harderian (lacrimal) gland of chicken. The study revealed that the whole-cell currents are dominated by outward rectifying currents which display slow inactivation times of the order of seconds. Records from excised outside-in and outside-out patches consistently revealed one channel type, a maxi-K channel. These maxi-K channels were shown to be both voltage and calcium sensitive. The single channel conductance of the maxi-K channel, with KCl solutions on both sides of the patch, ranged from 200–265 pS (n=26). Both whole-cell currents and single channel activity (outside-out) were reduced by the introduction of 10 mM TEA in the bath. The ease with which a large number of plasma cells can be isolated free of undifferentiated B-lymphocytes makes this preparation ideal for studying the relationship between the electrophysiological properties and immunoglobulin secretion in plasma cells.

Anti-muscarinic acetylcholine receptor-like immunoreactivity in lacrimal glands.

Lacrimal glands are extensively innervated by both the parasympathetic and sympathetic divisions of the autonomic nervous system. The neurotransmitters acetylcholine and norepinephrine are present in nerve fibers distributed among the secretory acini. Physiological studies using glands from a number of different species suggest that muscarinic acetylcholine receptors and both alpha and beta adrenergic receptors are present on the secretory acinar cells. There are no data, however, on the anatomic distribution of any neurotransmitter receptor in lacrimal glands. Given that the innervation density is sufficiently low so that it is unlikely that each acinar cell is directly innervated (see Walcott et al, this volume), it becomes even more important to determine the distribution of receptors in the glands in order to understand the control of lacrimal gland secretion.

Effects of anthrax lethal toxin on human primary keratinocytes

Aims:  To investigate the effects of anthrax lethal toxin (LeTx) on human primary keratinocytes.

Second Messenger Modulation of IgG Secretion from Chicken Lacrimal Gland

Avian tears originate from two sources: a relatively small lacrimal gland located at the lateral canthus of the eye, and a much larger Harderian gland located medial to the eyeball within the orbit. In the chicken, the Harderian gland is the dominant orbital gland1, producing a mucoid secretion2,3 that aids in corneal lubrication.

The Role of Membrane Channels in IgG Secretion by Plasma Cells in the Chicken Lacrimal Gland

One of the more ubiquitous cell types within the parenchyma of the lacrimal gland is the plasma cell. It is solely responsible for the production of immunoglobulins which are secreted into tears. While it is true that immunoglobulins such as IgA are modified by epithelial cells, it is still the plasma cell which acts as the source. In the case of IgG there is no involvement of the epithelium other than to act as a passive filter via the lateral interstitial space1. In terminally differentiated B-lymphocytes or plasma cells the secretion of immunoglobulin has been thought of as a constitutive-like process. The plasma cells simply secrete at a constant rate2. The processes which cause differentiation into plasma cells and hence increased output of immunoglobulin have been major focal points. It is thought that alteration in immunoglobulin output is affected more by changes in cell number rather than dynamic changes within individual cells’ secretory ability.

Pertussis composite nanofibrous membranes as an acellular transdermal whooping cough vaccine

Whooping Cough has globally resurfaced due to the suboptimal quality of the traditional vaccines, cyclic variations in its pattern and discovery of new strains of the causative agent, Bordetella pertussis. Our studies provide a proof of principle for the development of a novel, solid state vaccine to counter the disease by successfully immobilizing Pertussis Toxin (PT), which is 200 times larger than molecules traditionally delivered through the skin, in electrospun nanofibrous membranes of the polymer, polyvinylpyrrolidone (PVP). The transdermal delivery of the functional protein was verified using an in vitro assay utilizing Chinese Hamster Ovary (CHO) cells. The semiquantitative assay allowed us to estimate the extent of clumping of the adherent cells in the presence of functional PT from the basal media and homogenized constructs of EFT-200 human skin organotypic models (MatTek). The functionality of PT was compared to that of standard solutions of known concentrations as low as 6.25 ng/ml to quantify the findings. The successful delivery of biologically active PT through a model of uncompromised full thickness human skin indicates that the nanocomposite coating is a promising candidate for a novel transdermal vaccine, and may be employed in future strategies that would supplant traditional vaccination methods.

Confirmation of protective antigen functionality in an electrospun, nanofibrous membrane

The use of Bacillus anthracis as a bioterrorist agent, in 2001, resulted in the death of 5 individuals. The pathogenicity of anthrax is due to the proteolytic effects of one of its components: the enzyme lethal factor (LF). LFs substrate is located within the cytosol of cells and its cleavage results in a silencing of the interleukin-6 (IL-6) cytokine signaling pathway. The entry of LF into the cytosol is facilitated by a binding protein: protective antigen (PA). Proposed here is a nanofibrous membrane, containing encapsulated PA, that can function as a transdermal anthrax vaccine. This membrane is produced using the voltage-intensive process of electrospinning. To confirm retention of protein biological activity throughout the process, Mono Mac 6 cells, a human monocytic cell line, were treated with electrospun PA after being activated with lipopolysaccharide (LPS) and dosed with LF. A down regulation of IL-6 production, determined through an ELISA, was used as an indicator for PA function. Functionality of electrospun PA was confirmed and the amount of functioning PA deposited on a 4 mm × 4 mm silicon wafer substrate was quantified using the dose-dependent response between PA present and IL-6 down regulated.