Hollis R. Williams

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

ABSTRACT The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

Methyl-14C-glycinated hemoglobin as a substrate for proteases

Abstract A simple radioisotopic assay for the proteolytic enzyme activity with methyl- 14 C-glycinated bovine hemoglobin as the substrate has been developed. The radioactive hemoglobin was prepared by the reaction of hemoglobin with a water-soluble carbodiimide and methyl [ 14 C]glycinate in 5 M guanidine hydrochloride.

Human polymorphonuclear leukocyte collagenase and gelatinase: Comparison of certain enzymatic properties

Collagenase and gelatinase of human PMN leukocytes were separated by serial chromatography. The enzymes were shown to be similar in latency, activatability, chromatographic and electrophoretic behavior and the response to inhibitors. They recognize the same peptide linkage for cleavage, only each with a distinct difference in the effect caused by the secondary binding sites of the substrate molecules.

Expression and Immunoaffinity Purification of Human Inducible Nitric-oxide Synthase INHIBITION STUDIES WITH 2-AMINO-5,6-DIHYDRO-4H-1,3-THIAZINE

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37°C and contained 1.15 ± 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret λmax at 396 nm with a shoulder at 460 nm and contained 0.28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 ± 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Effects of human polymorphonuclear leukocyte collagenase on sub-component C1q of the first component of human complement.

Abstract The similarities in the structure and properties of C1q and collagen prompted us to examine the susceptibility of C1q to human polymorphonuclear leukocyte collagenase. Incubation of C1q with a collagenase preparation resulted in no change in (1) the binding of C1q to immunoglobulin aggregates, (2) the hemolytic function of C1q as measured by reconstitution of C1q-depleted serum in immune hemolysis, or (3) the structural properties of C1q as revealed by gel electrophorettic patterns of the whole molecule or its polypeptide chains. In contrast, rapid inactivation and degradation of C1q was caused by leukocyte elastase. The collagenase preparation was, however, capable of cleaving reduced and carboxamidomethylated C1q into discrete fragments. This activity was attributed to a gelatinase present in the enzyme preparation since (1) the cleavage reaction was inhibited by denatured collagen but not by native collagen and (2) a collagenase fraction free of gelatinolytic activity could not degrade reduced and carboxamidomethylated C1q, while a gelatinase fraction devoid of collagenase activity retained the capacity to effect reduced and carboxamidomethylated C1q. Both collagenase and gelatinase activities were activated from the latent form by trypsin, and inhibited by EDTA. Therefore, it appears that native C1q lacks the structural features present in collagen which are recognized by leukocyte collagenase for hydrolytic action even though the denatured molecule still contains that region capable of being cleaved by gelatinase.

Inhibition of the local hemorrhagic Shwartzman reaction by an acid proteinase inhibitor, pepstatin

Nachweis, dass das lokaleShwartzman-Phänomen in Kaninchen durch intradermale oder i.v. Gabe von Pepstatin, einem sauren Proteinase-Hemmer, oder von Sojabohnen-Trypsin-Hemmer kurz vor der auslösenden Injektion von bakteriellem Endotoxin unterdrückt werden kann.

PROTEOGLYCAN-DEGRADING ACTIVITY OF HUMAN STROMELYSIN-1 AND LEUKOCYTE ELASTASE IN RABBIT JOINTS. QUANTITATION OF PROTEOGLYCAN AND A STROMELYSIN-INDUCED HABR FRAGMENT OF AGGRECAN IN SYNOVIAL FLUID AND CARTILAGE

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.