Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes.

Sphingosine 1-phosphate (S1P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G protein-coupled receptors, S1P(1,2,3,4,5). Physiological effects of S1P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S1P(1), with IC(50) values for ligand binding between 0.3 and 14 nM. The correlation between S1P(1) receptor activation and the ED(50) for lymphocyte reduction was highly significant (p < 0.001) and lower for the other receptors. In contrast to S1P(1)-mediated effects on lymphocyte recirculation, three lines of evidence link S1P(3) receptor activity with acute toxicity and cardiovascular regulation: compound potency on S1P(3) correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S1P(1) relative to S1P(3); and toxicity, bradycardia, and hypertension were absent in S1P(3)(-/-) mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S1P(3) in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S1P(1) expression was restricted to the vascular endothelium.

CCR5, CXCR4, and CD4 are clustered and closely apposed on microvilli of human macrophages and T cells.

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.

Immunohistochemical localization of types 1 and 2 5alpha-reductase in human scalp.

The predominant form of 5alpha-reductase (5aR) in human scalp is 5aR1. None the less, clinical studies have shown that finasteride, a selective inhibitor of 5aR2, decreases scalp dihydrotestosterone and promotes hair growth in men with androgenetic alopecia. Immunolocalization studies were thus carried out to examine 5aR isozyme distribution within scalp and, in particular, to determine whether 5aR2 might be associated with hair follicles. 5aR2 was localized using both a rabbit polyclonal and a mouse monoclonal antibody. 5aR1 was detected with a mouse monoclonal antibody. The specificity of these reagents was demonstrated both by immunofluorescence and Western blot analyses of COS cells overexpressing human 5aR1 or 5aR2. When cryosections of scalp from men with androgenetic alopecia were stained with antibody against 5aR2, using immunoperoxidase avidin-biotin complex methodology, immunostaining was observed in the inner layer of the outer root sheath and, in more proximal regions of the follicle, in the inner root sheath. Staining was also prominent in the infundibular region of the follicle, with less intense staining extending throughout the granular layer of the epidermis. Some staining was also seen in sebaceous ducts. Similar results were obtained with both the polyclonal and monoclonal 5aR2 antibodies. In contrast, in scalp cryosections stained with antibody to 5aR1, no immunostaining was observed within hair follicles. Intense staining for the type 1 isozyme was, however, detected within sebaceous glands. Our immunolocalization data suggest that the results seen in clinical trials of men with male pattern hair loss treated with finasteride may be due, at least in part, to local inhibition of 5aR2 within the hair follicle.

Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.

OBJECTIVE: Two major cleavage sites, one mediated by metalloproteinases (MMPs) and the other by an as-yet unidentified enzyme termed aggrecanase, have been observed in aggrecan. To learn more about the relative contribution of these enzymes during cartilage degradation, this study assessed the occurrence of both specific neoepitopes in cartilage during murine arthritis and examined the correlation between neoepitope formation and different aspects of cartilage damage. METHODS: Reversible cartilage damage was induced in mice in the zymosan-induced arthritis (ZIA) model, partly irreversible cartilage damage in the antigen-induced arthritis (AIA) model, and irreversible, destructive cartilage damage in the collagen-induced arthritis (CIA) model. Immunolocalization techniques were used to detect the specific C-terminal neoepitopes VDIPEN (MMPS) and NITEGE (aggrecanase). RESULTS: In normal cartilage from young adult mice, no VDIPEN epitopes were detected, but a limited amount of NITEGE epitopes were already present. During the early phase of proteoglycan (PG) depletion, NITEGE expression was raised substantially in all arthritis models. VDIPEN epitopes were not detected in this early phase of cartilage destruction. When PG depletion progressed toward advanced cartilage damage, VDIPEN epitopes were induced. During ZIA, minimal induction of VDIPEN was observed, whereas in AIA, strong, but partly reversible, VDIPEN staining was evident, and in CIA, an extensive presence and persistence of the MMP-induced neoepitope was seen. When VDIPEN epitopes were intensely present, NITEGE epitopes were greatly reduced at that site in the cartilage. CONCLUSION: Presence of VDIPEN epitopes in cartilage correlated with severe cartilage damage, but these epitopes were not detected during early PG degradation. This suggests a limited role for VDIPEN-inducing MMPs in early PG degradation during murine arthritis. In contrast, aggrecanase epitopes were induced before the appearance of VDIPEN epitopes, but they disappeared with progression of cartilage damage.

Increased vulnerability of postarthritic cartilage to a second arthritic insult: accelerated MMP activity in a flare up of arthritis.

OBJECTIVE: Murine antigen induced arthritis (AIA) is a chronic, smouldering inflammation. Flares of arthritis can be induced by antigen rechallenge or exposure to inflammatory mediators like interleukin 1 (IL1). These flares are characterised by a fast and marked proteoglycan (PG) depletion if compared with the initial arthritis. This study investigated the involvement of metalloproteinases in both the initial and the flare phase of arthritis. METHODS: Murine AIA was induced and a flare up of arthritis was induced by injection of 10 ng of IL1beta. Messenger RNA levels of MMP-1 and -3 were studied by RT-PCR. MMP activity in cartilage, during both primary AIA as well as the flare up of arthritis, was studied by immunodetection of MMP specific neoepitopes in aggrecan (VDIPEN). Cartilage just before flare induction was analysed for presence of MMPs at the mRNA level as well as at the protein level by zymography. RESULTS: At the onset of AIA, a fast upregulation of mRNA for stromelysin and collagenase was noted. However, no VDIPEN epitopes were detected during this early phase of arthritis. They appeared when PG depletion was severe at day 7 of arthritis and disappeared when cartilage was repaired. IL1 injection into a knee joint at week 4 of AIA caused a flare up of arthritis, coinciding with a fast and marked PG degradation. This degradation was characterised by accelerated expression of VDIPEN epitopes if compared with the expression in primary AIA. Analysis of cartilage at week 4 of AIA showed still increased mRNA levels of MMP-1 and -3. Moreover, increased levels of latent MMPs were present as well, as APMA activation induced profound VDIPEN epitope. In vitro exposure to IL1 did show increased PG breakdown but no VDIPEN expression, suggesting that factors in addition to IL1 are needed to cause the in vivo VDIPEN expression. CONCLUSIONS: The fast and marked PG depletion seen in a flare up of AIA coincides with accelarated expression of MMP induced neoepitopes compared with expression during primary AIA. This accelerated expression is probably linked to increased levels of latent enzyme, which were found to be present in the cartilage before induction of a flare up.

Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease.

BACKGROUND & AIMS: Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied. METHODS: COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry. RESULTS: COX-1 protein was expressed at equal levels in normal, Crohn's disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohn's disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohn's colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohn's colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohn's ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase. CONCLUSIONS: COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.

Subcellular localization of prostaglandin endoperoxide H synthases-1 and -2 by immunoelectron microscopy.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs like aspirin and ibuprofen. These enzymes catalyze the committed step in the formation of prostanoids from arachidonic acid. Although PGHS-1 and -2 are similar biochemically, a number of studies suggest that PGHS-1 and PGHS-2 function independently to form prostanoids that subserve different cellular functions. We have hypothesized that these isozymes may reside, at least in part, in different subcellular compartments and that their compartmentation may affect their access to arachidonic acid and serve to separate the functions of the enzymes. To obtain high resolution data on the subcellular locations of PGHS-1 and -2, we employed immunoelectron microscopy with multiple antibodies specific to each isozyme. Both PGHS-1 and -2 were found on the lumenal surfaces of the endoplasmic reticulum (ER) and nuclear envelope of human monocytes, murine NIH 3T3 cells, and human umbilical vein endothelial cells. Within the nuclear envelope, PGHS-1 and -2 were present on both the inner and outer nuclear membranes and in similar proportions. Western blotting data showed a similar distribution of PGHS-1 and -2 in subcellular fractions, and product analysis using isozyme-specific inhibitors suggested that both enzymes generate the same products in NIH 3T3 cells. Thus, we are unable to attribute the independent functioning of PGHS-1 and PGHS-2 to differences in their subcellular locations. Instead, the independent operation of these isozymes may be attributable to subtle kinetic differences (e.g. negative allosteric regulation of PGHS-1 at low concentrations of arachidonate (500-1000 nM)). A further conclusion of importance from a cell biological perspective is that membrane proteins such as PGHS-1 and -2, which are located on the lumenal surface of the ER, are able to diffuse freely among the ER and the inner and outer membranes of the nuclear envelope.

Interleukin-1 receptor antagonist prevents expression of the metalloproteinase-generated neoepitope VDIPEN in antigen-induced arthritis.

OBJECTIVE: To investigate the relationship between occurrence of the matrix metalloproteinase-generated neoepitope VDIPEN and proteoglycan (PG) loss in arthritis, and to examine the role of interleukin-1 (IL-1) in VDIPEN expression. METHODS: VDIPEN expression was investigated in murine antigen-induced arthritis by immunolocalization studies on joint sections. The involvement of IL-1 in VDIPEN expression was studied by blocking of IL-1 using IL-1 receptor antagonist (IL-1Ra). RESULTS: Profound PG loss was evident early in arthritis, without significant VDIPEN expression. Full expression of the neoepitope appeared after a few days, when PG depletion was severe, and disappeared at late stages when cartilage showed recovery from PG depletion. At sites where chondrocyte death occurred and cartilage did not recover from the initial cartilage depletion, VDIPEN expression remained present. Prophylactic IL-1Ra treatment of arthritic mice resulted in almost complete prevention of VDIPEN expression. However, IL-1Ra had only a minor effect on PG depletion, emphasizing that there is no correlation between VDIPEN and early PG depletion. CONCLUSION: This study indicates that IL-1 is involved in VDIPEN expression. Although VDIPEN-inducing metalloproteinases do not seem to be involved in early PG depletion during antigen-induced arthritis, metalloproteinase neoepitopes are present when PG depletion is severe.

Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction.

OBJECTIVE: It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. METHODS: The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. RESULTS: SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. CONCLUSION: Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

Aggrecanase and metalloproteinase-specific aggrecan neo-epitopes are induced in the articular cartilage of mice with collagen II-induced arthritis.

OBJECTIVE: To analyze the roles of two classes of proteinases, 'aggrecanase', and matrix metalloproteinases (MMPs), in chondrodestruction during murine collagen-induced arthritis (CIA). METHODS: Generation of the 'aggrecanase' neo-epitope (NITEGE373), and the MMP neo-epitope (VDIPEN341) within aggrecan was studied by immunoperoxidase microscopy using specific anti-peptide antibodies in normal and stromelysin-1 (SLN-1) deficient knockout mice with CIA. RESULTS: High levels of NITEGE373 and VDIPEN341 neo-epitopes were observed in foci within CIA paw articular cartilage exhibiting depletion of glycosaminoglycans, in advance of significant cartilage erosion. The highest concentrations of NITEGE373 and VDIPEN341 labeling were observed and often co-distributed in the chondrocyte pericellular matrix, suggesting that stimulated chondrocytes can synthesize and/or activate both enzymes. Other regions of the cartilage frequently exhibited either NITEGE373 or VDIPEN341 labeling, but not both neo-epitopes simultaneously, suggesting that 'aggrecanase' and MMP cleavages of aggrecan may be generated independently. No detectable differences were observed in expression or distribution of either neo-epitope in SLN-1 knockout versus wild-type mice. In addition, in vitro digestion of joint sections with SLN-1 did not alter the expression of cartilage NITEGE373, while markedly increasing VDIPEN341 labeling. Peripheral nerves and brains of naive mice also exhibited intense anti-NITEGE373 labeling. CONCLUSIONS: These data indicate that NITEGE373 and VDIPEN341 aggrecan neo-epitopes are sensitive and specific markers of early joint pathology, and are consistent with the hypothesis that SLN-1 does not have 'aggrecanase' activity, and that 'aggrecanase' is distinct from the MMPs which cleave aggrecan at the MMP site.

Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease.

BACKGROUND & AIMS: Inducible nitric oxide synthase (iNOS) is generated in several cell types by treatment with lipopolysaccharides or cytokines. Earlier studies suggested that ulcerative colitis is associated with increased NO produced by iNOS; however, the cellular source of the NO synthesis was not identified. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide to produce peroxynitrite, which reacts with tyrosine to form nitrotyrosine in cellular proteins. METHODS: Using immunoperoxidase microscopy with a new monospecific human iNOS antibody (NO-53), the cellular distribution of iNOS and nitrotyrosine was examined using human colonic mucosa from normal bowel, ulcerative colitis, Crohn's disease, and diverticulitis. RESULTS: Intense focal iNOS labeling was localized to the inflamed colonic epithelium in ulcerative colitis, Crohn's disease, and diverticulitis but was not detectable in the uninflamed epithelium. Nitrotyrosine labeling was also observed in the inflamed colonic epithelium and was associated with nearby iNOS staining; nitrotyrosine was undetectable in normal mucosal epithelium. iNOS and nitrotyrosine were also detected in lamina propria mononuclear cells and neutrophils. CONCLUSIONS: These findings suggest that iNOS is induced in the inflamed human colonic epithelium and is associated with the formation of peroxynitrite and the nitration of cellular proteins.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

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.

5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.

5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.

5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes.

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

Optimal humanization of 1B4, an anti-CD18 murine monoclonal antibody, is achieved by correct choice of human V-region framework sequences.

The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.

Recombinant human interleukin-1 beta-induced increase in levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid.

OBJECTIVE: To evaluate the effects of intraarticular injection of recombinant human interleukin-1 beta (IL-1 beta) on levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid (SF), and to determine the effects of leukocyte depletion on SF proteoglycan and stromelysin levels. METHODS: Levels of leukocytes and of proteoglycans, stromelysin, and collagenase were evaluated 12 hours after the intraarticular injection of various doses of IL-1, and over a 24-hour period after injection at a single dose level. We used a monoclonal antibody (MAb) against leukocyte integrins, which markedly depressed leukocyte accumulation in SF, to evaluate the role of synovial leukocytes on IL-1-induced increases in SF proteoglycan and stromelysin levels. RESULTS: Levels of both proteoglycans and stromelysin increased in the IL-1-injected joints between 4 hours and 24 hours after the injection of a single 200-ng dose of IL-1. The highest levels of stromelysin and proteoglycans were achieved with IL-1 doses greater than or equal to 100 ng. Infiltration of polymorphonuclear cells (PMN) into the joint fluid of the IL-1-injected rabbits also increased, in a dose-dependent manner. Treatment of rabbits with MAb 1B4 markedly reduced infiltration of PMN into the joint, without affecting either stromelysin or proteoglycan levels. CONCLUSION: Taken together, the data suggest that there is a coordinate increase in SF stromelysin and proteoglycan levels in rabbits injected with IL-1, and that leukocytes play a minimal role in the accumulation of proteoglycans and stromelysin in the SF.

Enhanced attenuation of meningeal inflammation and brain edema by concomitant administration of anti-CD18 monoclonal antibodies and dexamethasone in experimental Haemophilus meningitis.

Antiinflammatory therapy has been shown to reduce the adverse pathophysiological consequences that occur in bacterial meningitis and to improve outcome from disease. In the present study, modulation of two principal steps of the meningeal inflammatory cascade was accomplished by concomitant administration of dexamethasone to diminish overproduction of cytokines in response to a bacterial stimulus and of a monoclonal antibody directed against adhesion-promoting receptors on leukocytes to inhibit recruitment of white blood cells into the subarachnoid space. Dexamethasone and antibody therapy produced a marked attenuation of all indices of meningeal inflammation and reduction of brain water accumulation after H. influenzae-induced meningitis in rabbits compared with results of each agent given alone and of untreated animals. In addition, the enhanced host's meningeal inflammatory reaction that follows antibiotic-induced bacterial lysis was profoundly ameliorated when dual therapy was administered without affecting clearance rates of bacteria from cerebrospinal fluid and vascular compartments. The combination of both therapeutic approaches may offer a promising mode of treatment to improve further the outcome from bacterial meningitis.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase induce reductase accumulation and altered lamellar bodies in rat forestomach keratinocytes.

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and a potent hypocholesterolemic agent, induces a hyperplastic thickening of the rat forestomach mucosa after oral administration of its active form, a hydroxyacid. We studied the effects of lovastatin on the intracellular accumulation of HMG-CoA reductase immunostaining and the accompanying morphological changes in rat forestomach keratinocytes by immunofluorescence microscopy and transmission electron microscopy (TEM). Administration of lovastatin hydroxyacid induced increases in HMG-CoA reductase levels within forestomach keratinocytes that were dose and time dependent and reversible. The adjacent glandular stomach epithelium did not exhibit induction of reductase. A pharmacologically inactive epimer of lovastatin hydroxyacid did not increase keratinocyte reductase accumulation, and lovastatin lactone induced minimal forestomach reductase. TEM of forestomachs from rats given lovastatin hydroxyacid demonstrated profound alterations in epidermal lamellar bodies (organelles that transport lipids and steroids to the intercellular spaces of the stratum corneum). Treated cells lacked internal lipid lamellae and failed to secrete sheets of lipid material into the intercellular spaces of the stratum corneum. We hypothesize that sustained inhibition of HMG-CoA reductase in rat forestomach keratinocytes induces accumulation of HMG-CoA reductase and hyperplasia by inhibiting sterol synthesis, assembly of lamellar bodies, and formation of intercellular lipid sheets.