This Article Abstract Full Text (PDF) All Versions of this Article: jhc.6A7070.2007v1 55/5/495    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Heale, C. E. Articles by Richardson, V. J. Search for Related Content PubMed PubMed Citation Articles by Heale, C. E. Articles by Richardson, V. J. Social Bookmarking                      What's this?

Progressive and Concordant Expression of PKC- and iNOS Phenotypes in Monocytes From Patients With Rheumatoid Arthritis: Association With Disease Severity

Catherine Elizabeth Heale, Goverdina Elisabeth Fåhræus-Van Ree, Proton Rahman and Vernon John Richardson

Faculty of Medicine (CEH,PR,VJR) and Department of Biology (GEF-VR), Memorial University, St John's, Newfoundland and Labrador, Canada, and Rheumatology Research, St. Clare's Mercy Hospital, St John's, NL, Canada (PR)

Correspondence to: Dr. Vernon John Richardson, Faculty of Medicine, Memorial University of Newfoundland, St. John's, NL, A1B3V6, Canada. E-mail: vrichard{at}mun.ca

	Summary

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Rheumatoid arthritis (RA) is a relatively common autoimmune disease with strong genetic and environmental determinants. The disease manifests itself as inflammation of the synovia and usually progresses to joint erosion and destruction. The disease can also be considered as a systemic disease because extra-articular manifestations are often observed throughout many organs and tissues of the body. Patients with severe RA have altered peripheral blood monocytes (PBM) that express activation markers. Two such markers, PKC- and iNOS, were studied using confocal laser scanning microscopy to determine how these markers are expressed during disease progression. Healthy individuals expressed neither of the two markers, but there was an elevated level of PKC- observed as the disease progressed (40% in mild RA and 100% in severe RA patients). Concordant expression of the two markers was observed in only 3% of PBM from mild RA patients, reaching 38% in severe RA patients. No cells expressing iNOS alone were observed in any of the patients studied. These data support the hypothesis linking PKC- expression with the regulation and predisposition to the development of the iNOS phenotype in severe RA patients. PKC- may therefore be a key regulator in the production of elevated plasma nitric oxide (NO) and corresponding circulating reactive nitrogen intermediates in severe RA and may be a possible target to regulate iNOS induction and NO production by monocytic cells in RA patients and possibly other inflammatory diseases. (J Histochem Cytochem 55:495–503, 2007)

Key Words: immunofluorescence • confocal microscopy • PKC- • iNOS • peripheral blood monocytes • rheumatoid arthritis

	Introduction

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
NITRIC OXIDE (NO) has been shown to have many beneficial physiological activities; however, overproduction of this molecule has been implicated in a number of pathologies including rheumatoid arthritis (RA). It has been demonstrated that NO is involved in the activation of metalloproteinases (Murrell et al. 1995) and acts as a key mediator of cellular apoptosis within the diseased joint (van't Hof et al. 2000). Patients with RA have elevated levels of synovial and serum nitrite and nitrate, both products of NO that are readily measurable (Farrell et al. 1992). Circulating NO products have also been implicated in the development of cardiovascular insensitivity and hypertension in RA patients (Bergholm et al. 2002). Patients with RA are also prone to an earlier and higher frequency of cardiovascular and cerebrovascular disease including accelerated atherosclerosis and abnormal endothelial function (Kaplan 2006).

Biosynthesis of NO is a complicated process under the control of three isoenzymes, nitric oxide synthases (NOS), namely, nNOS, eNOS, and iNOS (Andrew and Mayer 1999). nNOS is a neuronal, constitutive, Ca²⁺- and calmodulin-dependent enzyme involved with neurotransmission. eNOS is also Ca²⁺ and calmodulin dependent and constitutively produced, mainly by endothelial tissues, and is involved with the regulation of vascular tone and maintenance of blood pressure. Finally, iNOS, an inducible enzyme produced by many cells including monocytes is involved in a number of immunological functions that require production of large and sustained quantities of NO. This inducible enzyme is Ca²⁺ independent (Hobbs et al. 1999).

The second marker studied, protein kinase C-eta (PKC-), is one of 12 PKC isotypes found in cells (Newton 1995). They are serine–threonine kinases involved in cytoplasmic signaling and have been implicated in numerous regulatory functions. These kinases have a complex tissue distribution and often compete or oppose regulatory functions. PKC isotypes are classified into three major families that in some ways delineate the cytoplasmic conditions in which the molecule is most active. The classical PKC molecules (, ß1, ß2, and ) have been shown to be activated by phospholipids and diacylglycerol and require Ca²⁺ for activity. Novel PKC molecules (, , , , and µ) have also been shown to be activated by phospholipids and diacylglycerol but are Ca²⁺ independent. Atypical PKC molecules ( and ) have been shown to be activated by phospholipids (Gomperts et al. 2002).

Murine monocytic cells and cell lines have been the main model used to study the induction of iNOS and production of NO by cells (Xiu and Liu 1998; Szabo 2000). However, human monocytes and monocytic cell lines behave very differently from their murine counterparts in their ability to respond to activators such as endotoxin and proinflammatory cytokines. The ability of human monocytes to produce NO following induction of iNOS has had mixed success and, only under certain circumstances, have these cells readily shown this phenotype (Panaro et al. 2003). In many cases, researchers have been unable to induce iNOS in human monocytes and observe NO production (Scheemann et al. 1993).

A number of hypotheses have been proposed to explain this, one of which suggests that the lack of response is due to the absence of PKC- in human monocytic cells (Pham et al. 2003a). The signaling pathway from endotoxin through the TLR-4 receptor leads to the transcription of iNOS. The role of PKC- in this process has not yet been established, but we believe that it may be involved in the regulation of phenotypical development. In murine monocytic cells, PKC- has been shown to be present but absent in human monocytic cells. Also, human monocytic cells have been reported to be resistant to lipopolysaccharide (LPS) induction of iNOS and NO. Transfection of PKC- into a human monocytic cell line resulted in the subsequent sensitivity of these cells to LPS and production of NO (Pham et al. 2003a). Second, peripheral blood monocytes (PBM) from patients with inflammatory diseases such as RA, psoriatic arthritis, and anklyosing spondylitis were shown to be positive for both PKC- and iNOS, whereas cells isolated from healthy controls were negative (Pham et al. 2003b). This study used RT-PCR to determine the presence or absence of PKC- and iNOS and could not pinpoint the level of protein expression, frequency of expression, or cellular distribution of these two markers. These data were also unable to resolve if these markers were found individually in two different cell phenotypes or were concordantly expressed in a single cell phenotype.

The above-mentioned cell transfection studies and the clinical findings with PKC- led to the hypothesis that there may be a possible direct correlation between the progression of the two markers, and that the expression of PKC- may be essential for switching to the iNOS phenotype, thus influencing production of NO and the severity of the disease. This hypothesis appeared to be supported by the above clinical findings (Pham et al. 2003b).

The objectives of the present study were to determine the level of expression of the two markers (PKC- and iNOS) and to determine the relative proportions of the different cell phenotype(s) expressing the two markers by using dual-labeling immunofluorescence and confocal laser scanning microscopy.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Patient Selection
Institutional Human Investigation Committee approval and patient consent were obtained prior to the initiation of this study. Blood samples were obtained from three groups: five healthy volunteers, five mild RA patients, and 10 severe RA patients. All RA patients satisfied the American College of Rheumatology criteria for this disease diagnosis (Arnett et al. 1988). The two RA groups were separated on the criteria that the mild RA group had less than five effused joints, whereas the severe RA group had more than 10 effused joints. Both RA groups had active disease, despite a variety of therapeutic interventions. Laboratory investigators were blinded with respect to disease status of the RA subjects until all laboratory work was completed.

Determination of Plasma NO (Nitrite + Nitrate)
Plasma NO was determined using a previously published method (Pham et al. 2003b). Briefly, plasma was treated using Escherichia coli nitrate reductase (Boehringer Mannheim, GmbH; Mannheim, Germany) to convert nitrate into nitrite after deproteination nitrite was determined using Griess reagent. Plasma nitrite levels were determined using a sodium nitrite standard curve. Results are shown in Figure 1 . Statistical analysis was done using Graph Pad Instat software, and significance level was set at p=0.05.

View larger version (8K): [in this window] [in a new window]   Figure 1 Plasma nitric oxide NO (nitrite + nitrate) levels in healthy, mild (RA), and severe RA groups. Statistical analysis is shown in the text.

Morphological Staining and Counting of Monocytes
Cells fixed as above were stained with a few drops of Wright's stain, allowed to stand for 2 min, and then followed by addition of two drops of resolving buffer (phosphate buffer, pH 6.8) and allowed to stand an additional 6 min to complete the staining process. Cells were then washed with tap water. One hundred cells were counted for each patient. These were observed at x400 magnification and grouped morphologically.

Immunofluorescent Staining Technique
An indirect dual-labeling immunofluorescent technique was used with two different primary antibodies and secondary antibodies labeled with two different fluorochromes. First, 200 µl of blocking buffer containing 5% mouse serum was added to each well and left overnight at 4C. Wells were washed twice with PBS, followed by addition of 200 µl of PBS containing a mixture of goat polyclonal anti-PKC- (1:100) and rabbit polyclonal anti-iNOS (1:100). These were allowed to stand at room temperature for 6 hr. Both primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA; PKC- cat. #sc-215-G, iNOS cat. #sc-650). Wells were washed three times with PBS and once with distilled water to remove unbound primary antibody. Then 200 µl of a mixture of secondary antibodies was added to each well. The mixture contained Cy2-conjugated donkey anti-rabbit (1:100) for the detection of iNOS and Texas Red-conjugated donkey anti-goat (1:100) for the detection of PKC-. Both secondary antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA; cat. #711-225-152 and cat. #705-075-131, respectively). Chamber slides were left overnight at 4C. The following day, wells were washed five times with PBS to ensure removal of non-bound secondary antibody, and then slides were mounted using 9:1 glycerol/PBS mixture. To eliminate errors due to technical problems including autofluorescence, nonspecific antibody binding and possible cross-reactivity of antibodies, various methods, and specificity controls were performed. These controls included wells with no primary antibodies or no secondary antibodies, and also mixed antibodies to test for cross-reactivity of primary with secondary antibodies were done for both the specific antibodies and non-corresponding secondary antibody. All controls were done for healthy controls and each patient sample screened.

Confocal Laser Scanning Microscopy
Slides were examined using the blue argon laser (488 nm) and green HeNe laser (543 nm) of the Olympus Fluoview laser scanning equipment (FV300) connected to an Olympus upright microscope (BX50WI) at a total magnification of x400 and x600 (Olympus; Tokyo, Japan). All observations of the digital images at x400 were standardized with 100% laser intensity and PMT settings for Cy2 and Texas Red emissions at 517 and 679, respectively. Data from both laser images were obtained simultaneously. Estimation of phenotypes was done by randomly eye counting five fields of view. Double-negative cells were estimated from the same images that were adjusted for intensity to allow visualization of all cells.

Integrated Optical Density Measurement
To quantify the levels of expression of the two markers, integrated optical density (IOD) measurements were made from five separate random fields of view per patient sample. For each patient group there were 25 (healthy and mild RA) or 50 (severe RA) estimations of intensity for both markers. IOD scores were determined using Image Pro Plus software (Version 4.1; Media Cybernetics, Silver Spring, MD), and the comparative levels of PKC- and iNOS expression were calculated per field of view. Mean and standard error of the mean values (n) of cell counts for each group were 250.5 ± 1.92 (25) 249.5 ± 1.40 (25), and 252.5 ± 1.04 (50) for healthy, mild RA, and severe RA, respectively. Cell counts were not significantly different, eliminating the necessity for corrections for variations in cell numbers.

Graphs were prepared using Graph Pad Prism 4.00 software, and statistical analyses were performed using InStat 3.05 software, using one-way ANOVA; p values 0.05 were considered significant.

	Results

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Details of Study Patients
Table 1 summarizes the clinical data. Healthy volunteers were not statistically aged matched with the two patient groups; however, none had any signs of inflammatory disease or were taking any anti-inflammatory medications. The normally accepted values for erythrocyte sedimentation (ESR) range from 0 to 9 mm/hr. All mild and severe RA patients, except patient 74, were well above this range, indicating the severity of the inflammatory disease in these two groups. As patients randomly volunteered in this study, they were all on a variety of medications including methotrexate (MTX), corticosteroids (prednisone), and various non-steroidal anti-inflammatory drugs (NSAIDs) or disease-modifying anti-rheumatoid drugs (DMARDs). Two mild RA patients (71 and 74) were being treated with anti-cytokine therapies.

Monocyte Purity
Isolated leukocyte numbers were consistent for all samples tested with a mean ± SEM of 2.00 ± 0.44 x 10⁶ cells/ml. Percentages of adherent monocytes, as determined with Wright's stain, were also consistent with a mean ± SEM of 87.6 ± 3.0% of cells being monocytes and the remaining cells lymphocytes.

Immunofluorescence and Marker Expression
Results of the immunofluorescence studies showing the presence of the two markers, PKC- and iNOS, are shown in Figure 2 . Data confirmed that autofluorescence and background fluorescence were both within acceptable limits (Figures 2A and 2B). In addition, there was no cross-reactivity for either PKC- or iNOS primary antibodies and their non-corresponding secondary antibody (Figures 2C and 2D, respectively). Emission wavelength interference was also within acceptable limits using Texas Red and Cy2 fluorochromes. Figures 2E–2G show the immunofluorescence staining of PBM from a representative healthy volunteer stained for PKC-, iNOS, and an overlay of the two images, respectively, illustrating the cellular distribution and cellular phenotypes present. Figures 2H–2J show the immunofluorescence staining of PBM from a representative mild RA patient stained for PKC-, iNOS, and an overlay of the two images, respectively. Figures 2K–2M show immunofluorescence staining of PBM from a representative severe RA patient stained for PKC-, iNOS, and an overlay of the two images, respectively. It was easily determined, from image overlays, if cells contained either or both of the two markers by counting the cells stained red or green and, in the case of concordant expression, cells that appeared yellow. Results show the absence of staining for either of the two markers in the healthy volunteers, the presence of staining for PKC- in the mild RA patient group, and staining for both markers in the severe RA patient group. Figures 2N–2P are higher magnification images of cells from a severe RA patient showing PKC-, iNOS, and a combination of these two markers. They are not views of the same cell but do show the relative cellular distribution of the markers and, in Figure 2P, the variations in distribution between them. Both markers appeared to localize within the cellular cytoplasm (Figures 2N and 2O). In Figure 2P it appears that iNOS differs from PKC- in that it concentrates much more in oval vesicle-like structures within the cell cytoplasm, and these appear more abundant close to the cell surface.

View larger version (48K): [in this window] [in a new window]   Figure 2 Representative fluorescence micrographs of peripheral blood monocytes (PBM). Method and specificity controls: (A) Internal fluorescence control. (B) No primary antibody but both secondary antibodies and control. (C) Goat anti-PKC- primary with Texas Red-conjugated anti-rabbit secondary antibody. (D) Rabbit anti-iNOS primary with Cy2-conjugated anti-goat secondary antibody. Healthy: (E) PKC-, (F) iNOS, (G) colocalization of PKC- and iNOS. Mild RA: (H) PKC-, (I) iNOS, (J) colocalization of PKC- and iNOS. Severe RA: (K) PKC-, (L) iNOS, (M) colocalization of PKC- and iNOS. Severe RA higher magnification images: (N) PKC-, (O) iNOS, (P) colocalization of PKC- and iNOS.

View larger version (18K): [in this window] [in a new window]   Figure 3 Proportions of PBM cell phenotypes in healthy, mild RA, and severe RA groups.

View larger version (12K): [in this window] [in a new window]   Figure 4 Mean integrated optical density (IOD) readings in healthy, mild RA, and severe RA groups. Expression of (top) PKC- and (bottom) iNOS. Bars are means ± SEM, n=5 (healthy and mild RA), n=10 (severe RA). Statistical analysis is shown in the text.

View larger version (9K): [in this window] [in a new window]   Figure 5 Correlation of PKC- and iNOS expression by PBM. Log/log plot of 20 paired mean values for IOD of PKC- vs IOD iNOS for individuals in the healthy, mild RA, and severe RA groups.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

The appearance of these markers in PBM has not yet been reported to have any link to prognosis or therapeutic effectiveness of any medications. However, we have reported expression of PKC- also in patients with psoriatic arthritis and ankylosing spondylitis (Pham et al. 2003b), and it may also be that they are associated with a number of other inflammatory diseases. Elevated iNOS, associated with severe RA, was also accompanied by elevated levels of plasma NO. Although we have no direct evidence that iNOS was the only source of the NO produced, it obviously must have contributed to this.

PBM phenotypes shown in Figure 3 provide additional information to data from earlier studies using RT-PCR. We now see that there are a number of macrophage phenotypes present, their comparative proportions, and how these vary with disease severity. In severe RA, all cells were positive for PKC-, and 38% of cells expressed iNOS. From both mild and severe RA data it may be seen that iNOS is found only in cells concordantly expressing PKC-. Interestingly, there were no cells expressing the iNOS-only phenotype. These data further support the possible switching of PBM phenotype during the progression of the disease from mild to severe RA.

Level of expression of the two markers as shown by IOD measurements (Figure 4) shows a progressive increase in expression of both markers that is clearly associated with disease severity. Thus, it would appear that RA subjects with low iNOS all have mild RA, as compared with high iNOS producers who appear to have a severe RA phenotype. We do acknowledge, however, that a prospective rather than cross-sectional study is required to be absolutely confident regarding a cause and effect and the sequential or concomitant nature of these observations.

If we now look at the level of expression of the two markers, we can see in Figure 5 that there is a very strong correlation between them, suggesting that both are linked, and further supporting the possibility that PKC- does possibly influence the development of the iNOS phenotype.

Figure 6 illustrates two possible routes to explain the phenotypical development of PBM in RA. Observations from our study indicate that expression of iNOS appeared to take place only in cells that were also positive for PKC-, and that iNOS was never found alone in PBM isolated from any of the subjects studied. The appearance of the two markers was progressive and then concordant. All these observations support the hypothesis that PKC- may be required for iNOS induction in human PBM. These observations, therefore, support the phenotypical development illustrated in Figure 6 (Scheme 1) with the appearance first of a PKC--expressing phenotype followed a phenotype expressing both markers (PKC+/iNOS+) in patients with severe RA. Figure 6 (Scheme 2) would also require the presence of two distinct phenotypes: one expressing only PKC- and the other expressing only iNOS. The latter was never clearly observed in any of the patients studied.

View larger version (12K): [in this window] [in a new window]   Figure 6 Hypothesized schemes for the development of macrophage phenotypes observed.

If our hypothesis is correct and there is a link between PKC- expression and the appearance of iNOS in human monocytes, then it may be possible to delay or prevent the phenotypical changes taking place by using specific PKC- inhibitors. This would be an interesting means of testing the hypothesis if only there were more highly specific inhibitors on the market to actually use clinically to test this.

Conventional therapies for the treatment of RA (prednisone, DMARDs, and NSAIDs) did not appear to have any effect on the monocyte phenotypical changes leading to iNOS expression and NO production in this study. Assuming that the stages hypothesized for the phenotypical differentiation of monocytes were distinct and clear, it may be possible to investigate the conditions under which the phenotypical changes might occur. Obviously there are systemic changes occurring during disease progression that lead to the appearance of iNOS and elevation of plasma NO. It is very probable that the phenotypical changes observed in PBM may be related to the type or levels of proinflammatory cytokines released by cells and tissues of the body during disease progression. It may therefore be possible to delay or even prevent the phenotypical switching by using anti-cytokine therapies. Work to this end has already been initiated in this laboratory. Preliminary results indicate that the above hypothesis may possibly be correct. It will be of great interest to observe the effects that various anti-cytokine therapies may have on the two markers and NO production in these patients, and if these reduce cardiovascular problems and prevent hypotension developing in these patients.

	Acknowledgments

 
Funding was provided by the Faculty of Medicine, Memorial University of Newfoundland and Rheumatology Research, St Clare's Mercy Hospital, St John's, NL.

The authors thank Maureen Gallant, Karen Stapleton, and Michael Goldsworthy for technical assistance, and a special thanks to Yvonne Tobin for keeping track of patients and volunteers used in this study.

	Footnotes

 
Received for publication August 4, 2006; accepted January 5, 2007

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 

Andrew PJ, Mayer B (1999) Enzymic function of nitric oxide synthases. Cardiovasc Res 43:521–531[Abstract/Free Full Text]

Arias M, Rojas M, Zabaleta J, Rodriguez JI, Paris SC, Barrera LF, Garcia LF (1997) Inhibition of virulent Mycobacterium tuberculosis by Bcg^r and BCG^s macrophages correlates with nitric oxide production. J Infect Dis 176:1552–1558[Medline]

Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, et al. (1988) The American Rheumatism Association 1987 revised criteria for classification of rheumatoid arthritis. Arthritis Rheum 31:315–324[Medline]

Bergholm R, Leirisalo-Repo M, Vehkavaara S, Makimattila S, Taskinen MR, Yki-Jarvinen H (2002) Impaired responsiveness to NO in newly diagnosed patients with rheumatoid arthritis. Arterioscler Thromb Vasc Biol 22:1637–1641[Abstract/Free Full Text]

Farrell AJ, Blake DR, Palmer RM, Moncada S (1992) Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis 51:1219–1222[Abstract/Free Full Text]

Gomperts BD, Tatham PER, Kramer IM (2002) Signal Transduction. New York, Academic Press

Guerau-de-Arellano M, Huber BT (2002) Development of autoimmunity in Lyme arthritis. Curr Opin Rheumatol 14:388–393[CrossRef][Medline]

Hobbs AJ, Higgs A, Moncada S (1999) Inibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol 39:191–220

Kaplan MJ (2006) Cardiovascular disease in rheumatoid arthritis. Curr Opin Rheumatol 18:289–297[Medline]

Kerr JR, Cunniffe VS, Kelleher P, Coats AJ, Mattey DL (2004) Circulating cytokines and chemokines in acute symptomatic parvovirus B19 infection: negative association between levels of pro-inflammatory cytokines and development of B19-associated arthritis. J Med Virol 74:147–155[CrossRef][Medline]

Murrell GA, Jang D, Williams RJ (1995) Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun 206:15–21[CrossRef][Medline]

Newton AC (1995) Protein kinase C: structure, function, and regulation. J Biol Chem 270:28495–28498[Free Full Text]

Panaro MA, Brandonisio O, Acquafredda A, Sisto M, Mitolo V (2003) Evidence for iNOS expression and nitric oxide production in human macrophages. Curr Drug Targets 3:210–221

Pham TNQ, Brown BL, Dobson PRM, Richardson VJ (2003a) Protein kinase C-eta (PKC-) is required for the development of inducible nitric oxide synthase (iNOS) positive phenotype in human monocytic cells. Nitric Oxide 9:123–134[CrossRef][Medline]

Pham TNQ, Rahman P, Tobin YM, Khraishi MM, Hamilton SF, Alderdice C, Richardson VJ (2003b) Elevated serum nitric oxide levels in patients with inflammatory arthritis associated with co-expression of inducible nitric oxide synthase and protein kinase C- in peripheral blood monocyte-derived macrophages. J Rheumatol 30:123–134

Scheemann M, Schoedon G, Hofer S, Blau N, And Schaffner A (1993) Nitric oxide synthase is not a constituent of the antimicrobial armature of human mononuclear phagocytes. J Infect Dis 167:1358–1362[Medline]

Szabo C (2000) Pathophysiological roles of nitric oxide in inflammation. In Ignarro LJ, ed. Nitric Oxide Biology and Pathology. New York, Academic Press, 841–872

van't Hof RJ, Hocking L, Wright PK, Ralston SH (2000) Nitric oxide is a mediator of apoptosis in the rheumatoid joint. Rheumatology (Oxford) 39:1004–1008

Veale DJ, Ritchlin C, Helliwell PS (2005) Immunopathology of psoriasis and psoriatic arthritis. Ann Rheum Dis 64(suppl 2):103–105

Xiu W, Liu L (1998) Nitric oxide: from a mysterious labile factor, to the molecule of the Nobel Prize. Cell Res 8:251–258[Medline]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This Article Abstract Full Text (PDF) All Versions of this Article: jhc.6A7070.2007v1 55/5/495    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Heale, C. E. Articles by Richardson, V. J. Search for Related Content PubMed PubMed Citation Articles by Heale, C. E. Articles by Richardson, V. J. Social Bookmarking                      What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact