Advances in Scleroderma Research, 2002

By Carol Feghali, Ph.D., Research Assistant Professor of Medicine, University of Pittsburgh (originally published in Scleroderma Voice, 2003 #1)

The annual meeting of the American College of Rheumatology (ACR) was held in New Orleans, Louisiana, October 25–29, 2002. The research presented on systemic sclerosis (SSc) during the meeting included clinical studies, epidemiological studies, and basic research.

Reproduction in SSc

Figure 1. Fetal Microchimerism. Fetal cells enter the mother's blood via the placenta. If the fetus is a male, the fetal cells are identified by the Y chromosome.

The detection of chimeric cells in patients with SSc has resulted in extensive studies examining the role of pregnancies in the disease.

Fetal microchimerism refers to the presence of fetal cells which have persisted for many years in the blood and tissues of patients with SSc (Figure 1). For example, the male Y-chromosome can be identified in the blood of women with SSc who have had male children.

Fetal Cells Identified as T Lymphocytes

Investigators in Philadelphia, Penn., reported that fetal cells can be detected in both clinically affected and unaffected skin of patients with SSc. These cells were found to be T lymphocytes, which are a type of white blood cell (WBC) believed to be important in the development of SSc.

Fetal Cells Present in Women Who Have Never Been Pregnant

Investigators in Seattle, WA, have also detected male fetal cells in the blood of SSc and healthy women who have never had sons and those who have never had children, miscarriages, or abortions. The researchers suggest that the male cells may have been transferred to these women from their mothers during pregnancy after their mothers had a son, since all had one or more older brothers.

Speculation that Reproduction Is a Risk Factor for SSc

Thus there are many possible sources of chimeric cells. The presence of these fetal cells and the increased occurrence of SSc in women, especially during childbearing years, have led to the speculation that reproduction may be one of the risk factors for the development of SSc.

Brazil Study Suggests Pregnancy Not a Factor

A reproductive study from Brazil examined pregnancy factors, including the number of pregnancies and the number of abortions in 118 patients with SSc and 72 patients with rheumatoid arthritis (RA) using a questionnaire. Their results indicate that pregnancy does not result in an increased risk or a protective effect for RA or for either limited cutaneous or diffuse cutaneous SSc.

Important to Control Mother’s Blood Pressure During Pregnancy

Pregnancies in SSc usually result in the birth of healthy infants.

For SSc patients with kidney failure, pregnancy can be complicated. An investigator in Washington, D.C., examined the outcome of pregnancy in 10 patients with SSc affecting the kidney, five of whom remained on angiotensin converting enzyme (ACE) inhibitors for control of blood pressure during their pregnancy, and five of whom discontinued this type of drug.

All five patients who continued taking ACE inhibitors during pregnancy gave birth to healthy infants.

Among the five taken off ACE inhibitors during pregnancy, there were three healthy infants, two small infants, and one stillbirth.

The latter five patients received other medications for blood pressure control.

Patients with the worst pregnancy outcome were those whose blood pressure was not well controlled with other medications.

Swedish Data Inconclusive on Possible Relationship Between Pregnancy and SSc

Reproductive history of SSc patients in a Swedish population-based registry was examined by investigators from Durham, NC, and Stockholm, Sweden.

The registry included a hospital inpatient listing covering the period from 1964 to 1999 and a national fertility listing covering the years since 1925.

Twenty-four percent of SSc patients in the registry never had children (nulliparous). Nulliparity was associated with an increased risk for SSc.

One of the drawbacks of these data is that information on spontaneous and induced abortions was not available.

Another possible interpretation is that pregnancy may have a protective effect in scleroderma, which is the opposite of the theory proposed above by microchimerism investigators.

Alternatively, patients with SSc may have some degree of infertility after their diagnosis, although this was not found to be the case in a study presented last year involving 214 women with SSc from the Pittsburgh database. In this previous study, women with SSc did not have reduced fertility compared to those with rheumatoid arthritis (RA) or healthy control women.

Autoantibody Disease Markers

Figure 2. Antigen-Antibody Recognition. An antibody recognizes a specific antigen (antibody generator) and binds to it.

Autoantibodies are antibodies generated against self-proteins, also known as antigens (Figure 2). In most cases, these antigens are proteins found in the nucleus of normal cells.

Autoantibodies have been detected in the blood of patients with SSc and other connective tissue diseases (CTDs) such as systemic lupus erythematosus (SLE), polymyositis/dermatomyositis (PM/DM), and Sjögren’s Syndrome (SS). Some of these antibodies are strongly associated with SSc (that is, they seldom occur in other diseases), e.g. anti-topoisomerase I, anti-RNA polymerase, anti-Th/To, anti-RNP, and anti-centromere, and sometimes these antibodies are associated with a higher risk for a particular internal organ involvement.

For example, SSc patients with autoantibodies against topoisomerase I (Scl-70), a protein whose normal function is to unwind DNA prior to cell division, have a high frequency (60-70%) of lung fibrosis. The presence of anti-RNA polymerase antibodies is correlated with the complication of “renal crisis.”

Korean Researchers Identify New Autoantibody

Researchers in Korea recently identified a new autoantibody in the blood of patients with SSc which recognizes a protein known as DNA binding protein B (Dbp B).

This antibody was not specific to SSc since it was detected in the blood of 40% of SSc patients and 7% of healthy controls and individuals with other CTDs.

The antibody was found in a larger proportion of patients with diffuse cutaneous SSc (55%) than with limited cutaneous SSc (24%).

Japanese Researchers Detect Anti-Wa Antibody

Another rare antibody was described by researchers in Japan and called anti-Wa antibody. It was detected in the blood of patients with SSc, RA, and also other CTDs, but most frequently in SSc patients.

Centromere Regions Found to Be Different in SSc Patients

Antibodies against centromeres in the nucleus of cells are identified in the blood of some patients with SSc, especially those with limited skin thickening, but also in persons with SS, SLE, and certain cancers.

Two different groups of investigators, one in Oklahoma City, Okla., and one in Baltimore, Md., have found that specific regions in the centromere that are recognized by these antibodies are different in SSc patients than in those with other autoimmune diseases.

Australian Researchers Find SSc Antibody That May Inhibit Colon Contraction

A research group in Adelaide, Australia, examined the blood of SSc patients and healthy controls for the presence of antibodies that may prevent intestinal muscle contraction and thus contribute to intestinal malfunction.

When added to the colon (large intestine) of a mouse that had been stimulated to contract, the antibody from 7 of 9 SSc patients prevented the contraction of mouse colon.

Non-Antibody Disease Markers

Several additional markers of disease severity and internal organ involvement were described.

Type I Collagen in Blood of SSc Patients Correlates with Skin and Lung Involvement

Increased production of molecules accumulating around tissue cells, such as collagen, results in the thickening of skin and internal organs in patients with SSc.

Research conducted in Paris, France, showed that fragments of the most common type of collagen (type I) are increased in the blood of patients with SSc and correlate with the severity of skin and lung involvement.

CD44 May Mark Less Severe Fibrosis

Studies from Kanazawa, Japan, suggest that another molecule, cluster of differentiation 44 (CD44), is increased in the blood of SSc patients with limited compared with diffuse skin fibrosis.

In addition, patients with lung fibrosis had lower levels of CD44 in their blood than patients without lung involvement.

Thus CD44 may be a marker of less severe fibrosis in SSc.

CD44 promotes the rolling of WBCs along blood vessels. Its absence may reduce the ability of WBCs to escape from blood vessels and to initiate the inflammation that precedes fibrosis.

Reduced Ability to Prevent Blood Clotting in SSc

One of the initial problems in SSc is a reduction in oxygen level (hypoxia) in the skin and internal organs, which injures cells. Endothelial cells (the cells which line blood vessels) play an important role in this process.

Researchers from Toledo, Ohio, and Florence, Italy, conducted a study on endothelial cells in small blood vessels in the affected tissues of patients with SSc. They noted that these cells in patients with SSc have a reduced ability to prevent blood clotting compared with those of healthy individuals.

These results were supported by research conducted in Florence and Ancona, Italy, in which investigators measured blood levels of factors which cause breakdown of blood clots in SSc patients and found some to be higher and some to be lower compared with those in healthy controls.

These findings suggest that treatments targeting the blood clotting system may be of benefit in SSc.

The Role of Hypoxia in SSc

To examine the role of hypoxia, investigators from Switzerland, Italy, and Germany grew skin biopsy cells known as fibroblasts and exposed them to low oxygen (hypoxic) conditions. Hypoxic SSc fibroblasts had increased “expression” of a small group of genes involved in the production of collagen.

Another group from London, U.K., reported that an enzyme known as xanthine oxidoreductase (XOR) is increased in fibroblasts from the skin of SSc patients. XOR can bind to collagen and, under low oxygen conditions, can cause an increase in the levels of reactive oxygen species, which may in turn be responsible for the abnormally active fibroblasts in SSc.

Using Microarray Technology to Examine Gene Expression

Expression of genes was also examined by investigators in Houston, Texas, in fibroblasts grown from the skin of a pair of identical twins, one with SSc and the other healthy.

They used “microarray analysis,” which allows identification of genes that are “switched on” or “switched off.” Some genes were switched on in the skin of the patient with SSc, while others were switched off compared to the skin of the healthy twin.

They also showed that genes were detected at abnormal levels in both the affected and apparently unaffected skin of the SSc patient, indicating that apparently unaffected skin cells had begun the early stages of activation.

Investigators also used microarray technology to compare the genes expressed in SSc skin to those expressed in the skin of patients with a SSc-related disease known as sclero-myxedema. Some of the genes that are switched on in sclero-myxedema were the same as those switched on in scleroderma.

Newly Identified: Nephrogenic Fibrosing Dermopathy

Another disease related to SSc that has been identified recently is Nephrogenic Fibrosing Dermopathy (NFD). Patients with this disease develop skin features of SSc with involvement of deeper tissues (fasciitis) after being on kidney dialysis.

Investigators in Philadelphia, Pa., noticed that skin specimens from NFD patients had increased levels of proteins and cells also found to be elevated in the skin of SSc patients.

Fibroblasts grown from the skin of NFD and SSc patients both produced excessive amounts of collagen.

Progress in Identifying Markers for Lung Involvement

One of the ways to examine inflammation in the lung is to do a bronchoscopy, insert some fluid into the lung, and then, after several minutes, suck back the fluid for analysis. This procedure is called a bronchoalveolar lavage (BAL). The number of WBCs recovered in BAL fluid usually indicates the degree of inflammation in the lungs.

In research work from Baltimore, Md., investigators examined BAL fluid WBCs of patients with SSc and found that these cells were “active” or “turned on,” which may explain their role in lung inflammation.

In a study from Switzerland and Poland, investigators found that the levels of two products known to promote inflammation and fibrosis, leukotriene B4 (LTB4) and E4 (LTE4), respectively, were increased in the BAL fluid from the lungs of 30 patients with SSc who had lung involvement compared with 10 healthy non-smoking controls.

The levels of LTB4 and LTE4 were highest in SSc patients who had the most severe lung inflammation, thus suggesting they are markers of inflammation or they may be participating in the inflammation.

Treating SSc

Calcium deposits (calcinosis) in and under the skin are common in SSc, especially in patients with the limited cutaneous subtype (old term “CREST variant”).

Minocycline Appears to Be Effective in Treating Calcinosis

Researchers from Plymouth, England, examined the effect of 50 mg or 100 mg daily of the antibiotic minocycline on calcinosis in 9 patients with limited cutaneous SSc.

Minocycline was tested because of its ability to bind to calcium and remove it from the circulation (chelation) and because of its antibacterial properties which help prevent secondary infections.

Eight of the 9 patients studied showed improvement in calcinosis and finger ulcerations related to calcinosis as early as one month after initiation of treatment.

Deposits which decreased in size while on minocycline became more prominent after discontinuation of treatment.

Minocycline Appears Not to Be Effective in Treating Skin Thickening

The effect of minocycline on skin thickening was examined by investigators from Houston, Texas, Detroit, Mich., Milwaukee, Wis., and Farmington, Conn., in 31 patients with SSc.

Ten patients did not complete the study due to worsening of their disease; and in those who did finish the study, there was no beneficial effect of minocycline on skin thickening.

ECL Found Effective in Treating Soft-Tissue Calcification

A different approach was used by investigators in Tucumán, Argentina, who used extracorporeal lithotripsy (ECL) to treat soft tissue calcification in patients with SSc, juvenile dermatomyositis, and calcific tendinitis of the shoulder.

ECL is a procedure commonly used to break up kidney stones using sound waves. Each session lasted 40 minutes, and most SSc patients received only one session.

Deposits became smaller and disappeared two weeks after the treatment.

Benefit included resolution of pain and improvement of joint mobility, which began at 24 hours after treatment and lasted up to four years after the procedure. There were no adverse effects.

Follow-Up on KPAB Treatment Implies Lasting Benefit

A treatment study of aminobenzoate potassium (KPAB) was conducted about 10 years ago and included 146 patients with SSc; no improvement in skin thickening was found.

The same investigators in Salt Lake City, Utah, recently did a follow-up on the patients who had participated in the original study to determine their current status. Their data showed that patients who had received KPAB for at least 3 months had a lower death rate compared to patients who did not receive KPAB.

TNFa Treatment Creates Temporary Lupus-Like Symptoms

Drugs that block tumor necrosis alpha (TNFa), such as Etanercept and Infliximab, are used for the treatment of RA and other joint diseases. One of the side effects of these drugs is the development of a lupus-like disorder.

These agents were used to treat two patients in Baltimore, Md., who had both SSc and RA, both of whom subsequently developed a lupus-like disease that resolved when the TNF blockers were stopped.

Study Suggests Lung Benefit from Iloprost

Lung involvement in SSc predominantly occurs in the form of pulmonary fibrosis (PF) or pulmonary hypertension (PHT). The latter can be present with or without PF.

When PHT occurs without PF, it can be effectively controlled with drugs called epoprostenoids.

Studies conducted in London, England, suggested that patients with PHT secondary to PF also benefit from Iloprost, a related drug treatment.

Alefacept Shows Lung Benefits

Investigators in Baltimore, Md., tested the effect of the drug Alefacept on lung disease in SSc patients. Alefacept prevents the activation of T lymphocytes.

Patients were treated for 12 weeks with weekly intravenous infusions of Alefacept. After treatment, the investigators showed that the number of blood and BAL T lymphocytes and other inflammatory WBCs was reduced.

During the study period, lung function remained stable, neither improving nor deteriorating. Alefacept was well tolerated.

Follow-Up on Autologous Stem Cell Transplantation Shows Lung Disease Recurrence

An experimental treatment for SSc is autologous stem cell transplantation (ASCT). In this approach, “stem cells” are isolated from the patient’s blood and stored.

The patient is then given chemotherapy and total body irradiation, which eliminate the immune system cells.

The stored “stem cells” are then given back to the patient to reconstitute the immune system with new cells.

The theory is that a “normal” immune system will result, replacing the defective immune system which might have caused or perpetuated SSc.

A study examining lung inflammation and lung disease severity in patients with early, severe SSc who underwent ASCT suggests that these patients had temporary improvement in lung function.

Unfortunately, recurrence of lung disease occurred about one year after ASCT. Three of the six patients in the study had a relapse of their lung disease and the other three developed new lung inflammation (alveolitis) during follow-up.

Follow-Up on Prostaglandin Treatment for Pulmonary Fibrosis

Last year’s review of advances in SSc research included results of a study from Kawasaki, Japan, in which lipid microspheres containing prostaglandin E1 (PGE1) were used to treat pulmonary fibrosis (PF) in SSc patients. The results showed stabilization of lung disease.

Prostaglandins are usually used to decrease the narrowing of blood vessels (vasospasm) that occurs in Raynaud’s phenomenon.

In last year’s study, PF in SSc patients receiving PGE1 progressed more slowly than in those not treated.

This year, the same investigators reported the effect of blocking the prostaglandin E2 (PGE2) receptor in an animal model of lung disease in which lung fibrosis develops in mice treated with the chemotherapeutic agent bleomycin. Mice in which the PGE receptor was blocked had less lung inflammation than untreated mice.

Prostaglandin Treatment for Raynaud’s Needs Larger Study

Investigators in Milan, Italy, followed blood markers in 10 SSc patients to monitor the effect of prostaglandin E1 cyclodextrin (PGE1), which was used for the treatment of severe Raynaud’s phenomenon.

In addition to widening (dilating) blood vessels, PGE1 can also protect vascular endothelial cells.

The study results suggest that a maintenance dose of 60 µg once per month does not completely protect endothelial cells, and thus more frequent injections or higher doses may be more effective.

A study examining a larger number of patients is needed.

Mouse Model for Lung Fibrosis

Figure 3. Receptor-Ligand Interaction. A receptor located on the surface of a cell is recognized by a ligand (e.g. prostaglandins, TGFb). The receptor and the ligand fit like a lock and key. When the ligand binds the receptor, a signal is transmitted into the cell generating a ligand/ receptor-specific reaction.

Two difficulties in conducting research on SSc are the limited number of human skin samples and the lack of a good animal model for research.

One model used for lung fibrosis is obtained by delivering bleomycin, a drug used for treating some cancers, to the lungs of mice via the trachea.

In research presented by investigators in Chicago, Ill., the skin of mice was injected with bleomycin, causing WBCs to migrate into the skin from blood vessels.

Bleomycin resulted in activation of proteins which can increase collagen production. The effect was similar to that demonstrated in the laboratory using transforming growth factor beta (TGFb), which is known to be activated in a wide variety of fibrotic conditions.

In work from London, England, and Houston, Texas, investigators used another animal model for studying SSc. They generated mice missing the intact receptor for TGFb. A “receptor” is a protein found on the surface of cells which can bind its “ligand,” such as TGFb, and start a cascade of signals in the cell (Figure 3).

These mice develop skin and lung fibrosis, presumably because of constant activation of TGFb-dependent pathways by TGFb.

Endoglin Receptor Levels Tied to Skin Fibroblasts from Diffuse Cutaneous SSc Patients

In a research study involving multiple investigators from the U.K. and the U.S., levels of endoglin, another receptor for TGFb found on the surface of cells, was found to be increased in skin fibroblasts grown in the laboratory from diffuse cutaneous SSc patients.

The increased levels of endoglin may be a mechanism by which SSc fibroblasts try to block TGFb from promoting fibrosis.

Survival Rates for SSc Patients with Lung Transplants

In the case of severe lung involvement, one treatment option is lung transplantation if a matching donor lung is available.

Clinical information on 29 SSc patients who underwent lung transplantation in Pittsburgh, Pa., and Baltimore, Md., was compared to that of patients with idiopathic pulmonary fibrosis (IPF), a condition in which PF is present without an underlying connective-tissue disease (CTD).

SSc patients accounted for 4% of all lung transplant recipients. The SSc patients ranged in age from 26 to 58 years and had an average disease duration of 13 years. Thirteen patients had pulmonary fibrosis (PF), 6 had pulmonary hypertension (PHT), and 10 had both PF and PHT. Eighteen of the transplanted SSc patients had limited or no skin thickening, and 11 had diffuse skin thickening.

SSc patients had somewhat reduced survival at one year compared to IPF patients, but by three years after lung transplantation, the survival was similar (60%).

Epidemiology

Preferred Patient Support

When 25 patients in Leeds, England, were asked to respond to a questionnaire regarding the role of the SSc nurse in their care, 19 responded that their preferred method of support is individual discussions with their nurse regarding their disease, their medication, and their family.

Normal Tension Glaucoma in SSc Patients

Normal tension glaucoma (NTG) has been associated with cardiovascular and other vascular diseases.

Investigators in Paris, France, examined the occurrence of normal tension glaucoma in a prospective study of 44 consecutive SSc patients—13 with diffuse cutaneous SSc and 31 with limited cutaneous SSc—who had their disease for an average of 10 years.

An increased frequency of NTG was found in patients with SSc compared to 37 patients with osteoarthritis and may be due to vascular involvement.

Cancer Risk Not Found to Be Higher for SSc Patients; Breast and Lung Cancers Examined

Recent studies, including one of 490 patients from the Sclero-derma Registry presented last year, have shown that cancer risk is not increased in SSc patients.

During the 2002 ACR meeting, the features of breast and lung cancer in patients with SSc were examined using over 2500 SSc patients from the Pittsburgh, Pa., database.

Of 57 patients who had breast cancer, 69% had diffuse cutaneous SSc versus 31% with limited cutaneous SSc. The average age of SSc patients with breast cancer was 57, and the cancer diagnosis in the majority was made within three years of the onset of SSc.

Of 52 patients who had lung cancer, 35% were men and 78% were smokers. The average age of the patients was 61 years and the lung cancer was diagnosed an average of 16 years after the onset of SSc. Patients diagnosed with lung cancer had worsening shortness of breath prior to the cancer diagnosis, and thus increased shortness of breath in late stage SSc may be a clue to search for lung cancer.

SSc Survival by Race

Investigators from Baltimore, Md., examined survival by race in 1,166 SSc patients followed over a period of 11 years at one medical center.

Women accounted for 83% of the patients. Seventy-five percent were Caucasian, 19% were African-American, and 6% were of other races. Patients with diffuse cutaneous SSc comprised 36% of the study group.

At three years, African-Americans had a somewhat lower survival rate than Caucasians (78.6% vs. 82.2%).

Durometer Found Useful for Measuring Skin Thickening

The degree of skin thickening is most accurately measured in SSc patients using the modified Rodnan skin score (MRSS).

Investigators in Boston, Mass., tested the use of a durometer (a hand held digital measuring device) on patients with SSc by comparing the reproducibility of four measurements at each of nine skin sites.

The durometer was reliable and its results comparable when used for determining skin score obtained by different physicians. Less variability was obtained among physicians using the durometer than using the MRSS.

Durometers may be a useful tool for determining response to therapy in clinical trials involving multiple centers and examining physicians.

Patient Awareness and Involvement Are Crucial for Future Advances in Scleroderma Research

In the past year, international efforts have resulted in numerous advances in SSc research. New potential treatments have been reported, new animal models for SSc are being examined, and basic research into mechanisms underlying the cause of SSc has progressed.

Several of the new findings were feasible due to the generosity of patients with SSc and their willingness to provide blood samples and skin biopsies to conduct critical research to find the cause and cure of scleroderma.

Patient awareness and involvement is crucial for future advances in SSc research.