Jørgen Rygaard

Cancer invasion and tissue remodeling : cooperation of protease systems and cell types

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non‐neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI‐1 and MMPs) can be expressed by either the non‐neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non‐neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection.

To understand the importance of quorum sensing in chronic Pseudomonas aeruginosa lung infection, the in vivo pathogenic effects of the wild-type P. aeruginosa PAO1 and its double mutant, PAO1 lasI rhlI, in which the signal-generating parts of the quorum sensing systems are defective were compared. The rat model of P. aeruginosa lung infection was used in the present study. The rats were killed on days 3, 7, 14 and 28 after infection with the P. aeruginosa strains. The results showed that during the early stages of infection, the PAO1 double mutant induced a stronger serum antibody response, higher production of pulmonary interferon gamma, and more powerful blood polymorphonuclear leukocyte (PMN) chemiluminescence compared to its wild-type counterpart. On days 14 and 28 post-infection, significantly milder lung pathology, a reduction in the number of mast cells present in the lung foci, a reduced number of lung bacteria, and minor serum IgG and IgG1 responses but increased lung interferon gamma production were detected in the group infected with the PAO1 double mutant when compared with the PAO1-infected group. Delayed immune responses were observed in the PAO1-infected group and they might be associated with the production of virulence factors that are controlled by the quorum sensing systems. The conclusion of this study is that functional lasI and rhlI genes of P. aeruginosa PAO1 play a significant role during lung infection.

Reduced metastasis of transgenic mammary cancer in urokinase‐deficient mice

A prominent phenotype of plasmin deficiency in mice is reduced metastasis in the MMTV‐PymT transgenic breast cancer model. Proteolytically active plasmin is generated from inactive plasminogen by one of 2 activators, uPA or tPA. We now find that uPA deficiency alone significantly reduces metastasis >7‐fold in the MMTV‐PymT model. We studied a cohort of 55 MMTV‐PymT transgenic mice, either uPA‐deficient or wild‐type controls. Tumor incidence, latency, growth rate and final primary tumor burden were not significantly affected by uPA deficiency. In contrast, average lung metastasis volume was reduced from 1.58 mm3 in wild‐type controls to 0.21 mm3 in uPA‐deficient mice (p = 0.023). Tumor cell dissemination to brachial lymph nodes was also reduced from 53% (28/53) in wild‐type controls to 31% (17/54) in uPA‐deficient mice (p = 0.032). Mice without plasminogen display a severe pleiotropic phenotype. By comparison, spontaneous phenotypes are modest in uPA‐deficient mice, probably because they still have active tPA. We show that metastasis is strongly and selectively decreased in uPA‐deficient mice, suggesting that uPA‐directed antimetastatic therapy would be efficacious and have limited side effects.

Chronic Pseudomonas aeruginosa lung infection is more severe in Th2 responding BALB/c mice compared to Th1 responding C3H/HeN mice

The chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) is characterized by a pronounced antibody response and microcolonies surrounded by numerous polymorphonuclear neutrophils (PMN). Poor prognosis is correlated with a high antibody response to P. aeruginosa antigens. An animal model of this infection was established in two strains of mice: C3H/HeN and BALB/c, generally known as Th1 and Th2 responders, respectively, which were challenged with alginate‐embedded P. aeruginosa. Mortality was significantly lower in C3H/HeN compared to BALB/c mice (p<0.025). P. aeruginosa was cleared more efficiently in C3H/HeN mice and significantly more C3H/HeN mice showed normal lung histopathology (p<0.02), and we found significantly fewer microabscesses in C3H/HeN mice than in BALB/c mice (p<0.005). In supernatants from P. aeruginosa antigen and concanavalin A‐stimulated spleen cells from the two strains of mice, the interferon‐ (IFN‐) γ levels were higher, whereas IL‐4 levels were lower in C3H/HeN mice than in BALB/c mice. The implications of these findings for CF patients with chronic P. aeruginosa lung infection are discussed.

Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response

Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases. Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection. To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re‐infected with P. aeruginosa 14 days after the initial infection. Singly‐infected BALB/c mice, as well as non‐infected mice, were used as controls. Decreased mortality and milder lung inflammation in re‐infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly‐infected mice. The improved outcome in re‐infected mice correlated with changes in CD4 cell numbers. Surface expression of LFA‐1 on pulmonary CD4 cells was increased in re‐infected compared with singly‐infected mice. Moreover, resistance to re‐infection was paralleled by a shift towards a Th1‐dominated response and increased IL‐12 production. No significant increase in serum IgG was observed in the re‐infected mice. In conclusion, these results indicate a protective role for a Th1‐dominated response, independent of antibody production, in chronic P. aeruginosa lung infection in CF.

MCA Sarcomas Induced in scid Mice are More Immunogenic than MCA Sarcomas Induced in Congenic, Immunocompetent Mice

With the aim of studying possible T‐cell mediated selection of the cells in growing tumours, 108 mice of the C.B‐17 strain, either immunocompetent C.B‐17 mice or histocompatible immunodeficient C.B‐17 severe combined immune deficiency (scid) were treated with 3‐methylcholanthrene (MCA) in two different dosages. A total of 51 tumours were obtained, 44 of which were established as uncloned tumour cell lines, and used for further study. Tumour incidence correlated with carcinogen‐dosage in that more tumours developed in groups treated with a high MCA dose than in groups treated with a low MCA dose, but not with immune status of the tumour host. No significant difference in the level of MHC class I molecule expression was found between the two groups of tumours. The rate of rejection after transplantation to syngeneic immunocompetent hosts was significantly higher for the scid tumours than for the non‐scid tumours. The authors suggest that this reflects an immunoselection performed by T cells in the immunocompetent host in which the tumour originated, which has eliminated highly immunogenic tumour cells, leaving non‐immunogenic tumour cells to grow.

Urokinase Plasminogen Activator Is Localized in Stromal Cells in Ductal Breast Cancer

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade that facilitates cancer invasion through degradation of the extracellular matrix, and high levels of uPA in human breast cancer tissue correlate with poor prognosis. We previously found that, in ductal breast cancer, uPA mRNA is highly expressed by myofibroblasts surrounding invasively growing cancer cells. However, the localization of uPA protein has not been settled in the published literature. Because uPA is a secreted molecule, it could conceivably be localized differently from its mRNA. We have studied the localization of uPA immunoreactivity in detail. Twenty-five cases of invasive ductal carcinoma were analyzed with three different uPA antibody preparations, all of which gave an essentially identical stromal staining pattern. Using double immunofluorescence, we identified uPA immunoreactivity in myofibroblasts and macrophages in all cases examined. Additionally, in approximately half of the tumors, we saw uPA staining of endothelial cells. In 3 of the 25 cases, a small subpopulation of the cancer cells was uPA-positive. We conclude that uPA immunoreactivity is almost exclusively associated with stromal cells, which thus play a major role in generation of proteolytic activity in ductal breast cancer.

Interferon‐gamma (IFN‐γ) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats

In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF), we studied whether the inflammatory response could be altered by intraperitoneal treatment with recombinant rat interferon‐gamma (rrIFN‐γ). Rats were treated either before or after intratracheal challenge with P. aeruginosa embedded in alginate beads. Rats treated after challenge had a significant reduction in the severity of macroscopic lung inflammation compared with rats treated before challenge (P=0.004) and controls (P=0.003). The histopathology in controls was dominated by numerous polymorphonuclear leucocytes (PMN) (≥90%) surrounding the alginate beads like in CF. This could be caused by a Th2‐like response. In contrast, a complete shift to a chronic‐type inflammation dominated by mononuclear leucocytes (≥90% lymphocytes and plasma cells) and granulomas was observed in both rrIFN‐γ‐treated groups of rats. This could be caused by a Th1‐like response. There was no significant difference in lethality between the groups, and the antibody titres against P. aeruginosa sonicate and alginate were similar in the treated rats and controls. Since the ongoing lung tissue damage in CF patients has been shown to be caused by elastase secreted by PMN, which dominate the P. aeruginosa lung infection, our findings offer a possible new strategy of modifying the inflammatory response in CF patients.

Ginseng treatment reduces bacterial load and lung pathology in chronic Pseudomonas aeruginosa pneumonia in rats.

The predominant pathogen in patients with cystic fibrosis (CF) is Pseudomonas aeruginosa, which results in a chronic lung infection associated with progressive pulmonary insufficiency. In a rat model of chronic P. aeruginosa pneumonia mimicking that in patients with CF, we studied whether the inflammation and antibody responses could be changed by treatment with the Chinese herbal medicine ginseng. An aqueous extract of ginseng was injected subcutaneously, and cortisone and saline were used as controls. Two weeks after challenge with P. aeruginosa, the ginseng-treated group showed a significantly improved bacterial clearance from the lungs (P < 0.04), less severe lung pathology (P = 0.05), lower lung abscess incidence (P < 0.01), and fewer mast cell numbers in the lung foci (P < 0.005). Furthermore, lower total immunoglobulin G (IgG) levels (P < 0.01) and higher IgG2a levels (P < 0.025) in serum against P. aeruginosa sonicate and a shift from an acute type to a chronic type of lung inflammation compared to those in the control and cortisone-treated groups were observed. These findings indicate that ginseng treatment of an experimental P. aeruginosa pneumonia in rats promotes a cellular response resembling a TH1-like response. On the basis of these results it is suggested that ginseng may have the potential to be a promising natural medicine, in conjunction with other forms of treatment, for CF patients with chronic P. aeruginosa lung infection.

Early immune response in susceptible and resistant mice strains with chronic Pseudomonas aeruginosa lung infection determines the type of T‐helper cell response

Most cystic fibrosis (CF) patients become chronically infected with Pseudomonas aeruginosa in the lungs. The infection is characterized by a pronounced antibody response and a persistant inflammation dominated by polymorphonuclear neutrophils. Moreover a high antibody response correlates with a poor prognosis. We speculated that a change from this Th2‐like response to a Th1‐like response might decrease the lung inflammation and thus improve the prognosis in CF patients. To investigate this, we infected C3H/HeN and BALB/c mice intratracheally with P. aeruginosa. In addition, we studied the early immune response leading to different Th responses. Mortality was lower in the C3H/HeN mice (p<0.005), they cleared the bacteria faster (day 3 p<0.01, day 7 p<0.02), had a milder lung inflammation (day 7 p<0.01, day 14 p<0.0005) and had a Th1‐like IgG subclass switch. At day 3, the C3H/HeN mice produced less NO and TNF‐α, (p<0.01 and p<0.03) and had the lowest IL‐10/IL12 ratio (p<0.05). At day 7, the C3H/HeN mice had the highest IFN‐γ (p<0.02), and the lowest IL‐4 (p<0.02) production in the lungs. In conclusion, these results show that the Th1‐reacting C3H/ HeN mice with chronic P. aeruginosa lung infection have a better disease outcome compared to the Th2‐reacting BALB/c mice, indicating that a Th1 response might be beneficial in CF patients with chronic P. aeruginosa lung infection.