Johanna Lönn

The Role of Interleukin-6 in Lipopolysaccharide-Induced Fever by Mechanisms Independent of Prostaglandin E2

Fever has been shown to be elicited by prostaglandin E(2) (PGE(2)) binding to its receptors on thermoregulatory neurons in the anterior hypothalamus. The signals that trigger PGE(2) production are thought to include proinflammatory cytokines, such as IL-6. However, although the presence of IL-6 is critical for fever, IL-6 by itself is not or only weakly pyrogenic. Here we examined the relationship between IL-6 and PGE(2) in lipopolysaccharide (LPS)-induced fever. Immune-challenged IL-6 knockout mice did not produce fever, in contrast to wild-type mice, but the expression of the inducible PGE(2)-synthesizing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase-1, was similarly up-regulated in the hypothalamus of both genotypes, which also displayed similarly elevated PGE(2) levels in the cerebrospinal fluid. Nevertheless, both wild-type and knockout mice displayed a febrile response to graded concentrations of PGE(2) injected into the lateral ventricle. There was no major genotype difference in the expression of IL-1beta and TNFalpha or their receptors, and pretreatment of IL-6 knockout mice with soluble TNFalpha receptor ip or intracerebroventricularly or a cyclooxygenase-2 inhibitor ip did not abolish the LPS unresponsiveness. Hence, although IL-6 knockout mice have both an intact PGE(2) synthesis and an intact fever-generating pathway downstream of PGE(2), endogenously produced PGE(2) is not sufficient to produce fever in the absence of IL-6. The findings suggest that IL-6 controls some factor(s) in the inflammatory cascade, which render(s) IL-6 knockout mice refractory to the pyrogenic action of PGE(2), or that it is involved in the mechanisms that govern release of synthesized PGE(2) onto its target neurons.

Lipoxin A4 Inhibits Porphyromonas gingivalis-Induced Aggregation and Reactive Oxygen Species Production by Modulating Neutrophil-Platelet Interaction and CD11b Expression

ABSTRACT Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A4 (LXA4) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA4 on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA4. Furthermore, we found that LXA4 significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA4 was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA4 antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

Cytokines and chemokines are differentially expressed in patients with periodontitis: possible role for TGF-β1 as a marker for disease progression.

Periodontitis is a chronic inflammatory disease characterized by destruction of periodontal tissue ultimately leading to bone destruction and has been associated with other inflammatory diseases, such as atherosclerosis. Attachment loss of periodontal tissue is primarily caused by host cell-derived immune responses against subgingival biofilm. The aim of the present study was to determine the cytokine profile in serum, saliva and gingival crevicular fluid (GCF) patients with periodontitis and healthy controls. We show that periodontitis patients exhibit higher numbers of periodontal pathogens and their immune responses are significantly altered. The levels of IL-6 in saliva and GCF were significantly suppressed, and while CXCL8 was not altered in serum, its expression levels were significantly suppressed in saliva and elevated in GCF. The T-cell-derived cytokine IL-2 did not differ between patients and controls in serum and saliva, but there was a significant suppression in GCF of patients. Interestingly, TGF-β1 levels were significantly elevated in serum, saliva and GCF in patients compared to controls. Furthermore, by using cultured gingival fibroblasts stimulated with wild type and proteinase mutant strains of Porphyromonas gingivalis, we show that the suppression of CXCL8 and IL-6, and the induction of TGF-β1 is primarily mediated by the proteolytic activity of lysine-specific proteinases. These results indicate that P. gingivalis is a major contributor to the altered immune responses and the pathology of periodontitis. Furthermore, the ease of sampling and analyzing cytokine expression profiles, including TGF-β1, in saliva and GCF may serve to predict the progression of periodontitis and associated systemic inflammatory diseases.

High Concentration but Low Activity of Hepatocyte Growth Factor in Periodontitis

BACKGROUND High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biologic activity requires binding to its receptors, the proto-oncogene c-Met and heparan sulfate proteoglycan (HSPG). This study investigates HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis. METHODS Saliva, gingival crevicular fluid (GCF), and blood samples from 30 patients with severe periodontitis and 30 healthy controls were analyzed for HGF concentration using enzyme-linked immunosorbent assay and binding affinity for HSPG and c-Met using surface plasmon resonance. The regenerative effects of saliva from three patients and controls were analyzed in an in vitro model of cell injury. Subgingival plaques were analyzed for the presence of 18 bacterial species. RESULTS Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P <0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P <0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared with controls, patients had a higher prevalence of periodontally related bacteria. CONCLUSIONS Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biologic activity at local inflammation sites was reduced, and this effect was associated with the amount of periodontal bacteria. Loss of function of healing factors may be an important mechanism in degenerative processes in periodontally susceptible individuals.

Fibroblast Growth Factor 23, Hepatocyte Growth Factor, Interleukin‐6, High‐Sensitivity C‐Reactive Protein and Soluble Urokinase Plasminogen Activator Receptor. Inflammation Markers in Chronic Haemodialysis Patients?

Sera from 84 haemodialysis (HD) patients and 68 healthy blood donors were analysed with commercially available ELISA techniques for fibroblast growth factor 23 (FGF‐23), hepatocyte growth factor (HGF), interleukin‐6 (Il‐6), high‐sensitivity C‐reactive protein (hs‐CRP) and soluble urokinase plasminogen activator receptor (suPAR), to find a possible correlation of FGF‐23 and HGF with the earlier recognized inflammatory markers Il‐6 and hs‐CRP or suPAR. All patients studied had significantly elevated levels of FGF‐23, HGF, hs‐CRP and suPAR as compared to the controls. Il‐6 and hs‐CRP correlated for patients (R = 0.6) as well as for patients and controls altogether. Ln (natural logarithm) of HGF correlated weakly with Ln Il‐6 and Ln CRP (R 0.28–0.37). Ln FGF‐23 correlated only with Ln HGF (r = −0.25) in controls. Ln HGF correlated with ln suPAR (r = 0.6) in both patients and controls. Although elevated as compared to controls, we found no correlation of FGF‐23 with the recognized inflammatory markers Il‐6, hs‐CRP, nor HGF or the new marker suPAR in HD patients. Ln HGF correlated with Ln Il‐6, Ln CRP and Ln suPAR. Although probably involved in vessel disease, FGF‐23 and HGF may play other roles than acting in inflammatory vessel disease in HD patients. Further studies are necessary to evaluate the role of these immunological markers in chronic haemodialysis patients with atherosclerosis.

An Antithrombin III product containing biologically active hepatocyte growth factor may be beneficial in deep ulcer infections

BACKGROUND Widely studied for the past 20 years, hepatocyte growth factor (HGF) has been identified as a regenerative marker and an important factor in the development and healing of injuries. Antithrombin III (AT III) is a protein in the blood stream with anti-thrombotic and anti-inflammatory properties and has been used as an adjuvant treatment along with antibiotics in severe sepsis. OBJECTIVE To study the content and properties of HGF in plasma-derived AT III products, and the regenerative effect in severe deep ulcer infections. METHODS Commercial AT III products were analyzed for the presence and biological activity of HGF. One AT III product containing biologically active HGF was used to treat 18 cases of critical, deep ulcer infections scheduled for major invasive intervention. The patients were followed up for 6-60 months. RESULTS The AT III products contained HGF with different biological activity. No adverse reactions were observed after local administration of AT III during the study or follow-up period. In 16 of 18 cases no surgical intervention was needed within the first 6 month of inclusion. CONCLUSION AT III products containing biologically active HGF may contribute to regeneration and healing in severe deep ulcer infections which do not respond adequately to different combinations of antibiotics alone.

Evaluation of hepatocyte growth factor as a local acute phase response marker in the bowel: The clinical impact of a rapid diagnostic test for immediate identification of acute bowel inflammation

BACKGROUND There are no rapid tests that can distinguish contagious gastroenteritis, which requires isolation at its onset, from exacerbation of chronic inflammatory bowel disease (IBD) or bowel engagement in the course of systemic inflammatory response syndrome (SIRS). Hepatocyte growth factor (HGF) is an acute phase cytokine that is produced at the site of injury. It has high affinity to sulfated glycan, and this binding affinity is lost during chronic inflammation. The fecal pH strongly impacts the prognosis for severe bowel disease. We developed a strip test to evaluate HGF as a local acute phase response marker in the bowel. This test assessed the binding affinity of HGF to sulfated glycans in fecal samples and determined fecal pH as an indicator of illness severity. METHODS Fresh feces from patients with diarrhea (n=513) were collected and tested blindly, and information about patient illness course and outcome was collected. Patients were classified based on the focus of inflammation and the cause of the symptoms. Objectively verified diagnoses of infectious gastroenteritis (n=131) and IBD onset/exacerbation and bowel cancer (n=44) were used to estimate the performance of the test strip. ELISA was performed on 101 freeze-thawed feces samples to determine the fecal HGF levels. RESULTS The test rapidly distinguished infectious gastroenteritis from non-infectious inflammatory causes of diarrhea (sensitivity, 87.96%; specificity, 90.9%; positive predictive value, 96.6%; negative predictive value, 71.4%; accuracy, 89.1%). Fecal pH (p<0.0001) and mortality within 28days of sampling (p<0.04) was higher in patients with sepsis/SIRS and diarrhea. The concentration of HGF was higher in strip test-positive stool samples (p<0.01). CONCLUSIONS HGF is a good local acute phase response marker of acute bowel inflammation. Test-strip determination of the binding affinity of fecal HGF to sulfated glycan was a rapid, equipment-free way to assess patients with diarrhea and to guide the diagnostic and therapeutic approaches on admission.

Hepatocyte growth factor in patients with coronary artery disease and its relation to periodontal condition

Hepatocyte growth factor (HGF) is an angiogenic, cardioprotective factor important for tissue and vascular repair. High levels of HGF are associated with chronic inflammatory diseases, such as coronary artery disease (CAD) and periodontitis, and are suggested as a marker of the ongoing atherosclerotic event in patients with CAD. Periodontal disease is more prevalent among patients with CAD than among healthy people. Recent studies indicate a reduced biological activity of HGF in different chronic inflammatory conditions. Biologically active HGF has high affinity to heparan sulfate proteoglycan (HSPG) on cell-membrane and extracellular matrix. The aim of the study was to investigate the serum concentration and the biological activity of HGF with ELISA and surface plasmon resonance (SPR), respectively, before and at various time points after percutaneous coronary intervention (PCI) in patients with CAD, and to examine the relationship with periodontal condition. The periodontal status of the CAD patients was examined, and the presence of P. gingivalis in periodontal pockets was analyzed with PCR. The HGF concentration was significantly higher, at all time-points, in patients with CAD compared to the age-matched controls (P< 0.001), but was independent of periodontal status. The HGF concentration and the affinity to HSPG adversely fluctuated over time, and the biological activity increased one month after intervention in patients without periodontitis. We conclude that elevated concentration of HGF but with reduced biological activity might indicate a chronic inflammatory profile in patients with CAD and periodontitis.

Sclerostin, TNF-alpha and Interleukin-18 Correlate and are Together with Klotho Related to Other Growth Factors and Cytokines in Haemodialysis Patients

Patients with chronic renal failure are known to have renal osteodystrophy (bone disease) and increased calcification of vessels. A new marker of bone disease, sclerostin, the two pro‐inflammatory cytokines tumour necrosis factor‐alpha (TNF‐alpha) and interleukin‐18 (IL‐18), and the fibroblast growth factor‐23 (FGF‐23) receptor‐associated marker Klotho were tested in 84 haemodialysis (HD) patients and in healthy controls. The patients had significantly higher levels of the three former markers than of the controls while Klotho was significantly higher in the controls. Low level, but significant, correlations were observed in the patient group when the levels of these four markers were compared to each other and to those of 5 cytokines and growth factors tested earlier; high‐sensitive CRP (hsCRP), interleukin‐6 (IL‐6), hepatocyte growth factor (HGF), fibroblast growth factor‐23 (FGF‐23) and soluble urokinase plasminogen activator (suPAR). Ln sclerostin correlated positively to Ln hsTNF‐alpha, Ln HGF and Ln suPAR. Ln hsTNF‐alpha correlated positively to Ln sclerostin, Ln hsCRP, Ln IL‐6, Ln FGF‐23, Ln suPAR and Ln IL‐18. Ln IL‐18 correlated positively to Ln suPAR and Ln TNF‐alpha. Ln Klotho correlated negatively to Ln hsCRP but did not correlate to Ln FGF‐23. The markers studied here may be involved in the calcification of vessels seen in HD patients due to a combination of inflammation and bone disease. The mechanisms are still not fully known but may be of importance for future therapeutic possibilities in this group of patients.

Lipoprotein modifications by gingipains of Porphyromonas gingivalis

Abstract Background and Objective Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low‐density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro. Material and Methods Plasma lipoproteins were isolated from whole blood treated with wild‐type and gingipain‐mutant (lacking either the Rgp‐ or Kgp gingipains) P. gingivalis by density/gradient‐ultracentrifugation and were studied using 2‐dimensional gel electrophoresis followed by matrix‐assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis‐induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid‐reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation. Results Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB‐100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high‐density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation. Conclusion Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.