Lisa McKinney
Biology 303 Section 501
Dr. Ely
November 8, 2007
Autoimmune diseases are very prevalent in the United States today with approximately 23.5 million sufferers. There have been eighty to one hundred autoimmune diseases identified which are chronic and life-threatening. Some examples of autoimmune diseases are psoriasis, Crohn’s disease, lupus, rheumatoid arthritis, scleroderma, Guillan Barre Syndrome and many others (Autoimmune Disease). The diseases can be systemic, affecting many organs, or localized, damaging only one organ. An autoimmune disorder occurs when the body produces an immune response against its own body tissues, which it fails to recognize as its own. Therefore, it categorizes those tissues as ‘foreign.’ The body then produces antibodies against its own tissues. These antibodies are called auto-antibodies (Autoimmune Disorders). The body follows a cascading reaction to fight a disease. Part of that reaction is inflammation. In autoimmune disorders, the inflammation is unnecessary and damages the body. The inflammation is the main cause of pain in the autoimmune diseases.
Psoriasis vulgaris is an autoimmune disease that Haider et al.examined. Psoriasis vulgaris is one of many types of Psoriasis, and affects the skin causing patches of red, itchy thickened skin. It is caused by a T-cell dependent inflammatory process in the skin which accelerates growth of epidermal and vascular cells in the psoriasis lesions (Krueger). Psoriasis and other autoimmune disease are treated with anti-TNF drugs.
Tumor necrosis factor (TNF) is a type of cytokine that affects lipid metabolism, coagulation and insulin resistance as well as affecting the function of endothelial cells in the lining of blood vessels (Medical Dictionary). A cytokine is a small protein released by cells with a specific effect on the interactions between cells. Cytokinesaffect the communication and behavior of cells; the cells include interleukins and cell signal molecules such as TNF and interferon’s (IFN’s) whose function is to trigger inflammation. These cytokines are the cause of inflammation in autoimmune diseases (Medical Dictionary).
Anti-TNF and anti-IFN drugs block the cytokines to prevent the inflammation and associated symptoms that area part of autoimmune disease. There are two common cytokine inhibitors. The first produces anti-TNF antibodies, infliximab, brand named Remicade and the second cytokine inhibitor produces anti-IFN antibodies and is called fontolizumab (Medical Dictionary). These cytokine inhibitors do not cure the autoimmune disease, but they do stop the inflammation and the symptoms that come as a result of the inflammation. These treatments are shown to have a wide range of serious side effects.
Not all of the various inflammatory molecules affected by the cytokine inhibitors are understood. The purpose of the study by Haider et al was to clarify how gene expression is regulated by blocking TNF or IFN by neutralizing antibodies in mixtures of activated mononuclear leukocytes. The study used peripheral blood mononuclear cell’s (PBMC’s) for their mixtures; these are mixtures of monocytes and lymphocytes from which the granulocytes have been separated out and removed (Lackie). Haider et al. believe that this mixture reflects actual tissues in autoimmune diseases.
The first part of the study involved the analysis of genes induced by T-cell activation (T-cells activated like they were fighting an infection) in PBMC’s versus purified T-cells (T-cells in normal healthy blood). Haider et al compared PBMC stimulation in various conditions of non-stimulated cells, blocking the IFN with fontolizumab and blocking TNF by infliximab. For the mixtures with the activated T-cells there was a strong response in cells whose function involved the induction of apoptosis. The data did not show a significant difference between the activated T-cells and the purified T-cells. This data is shown in figure 1:
This figure shows the various conditions that the PBMC’s are tested for the reaction of the gene expression. The dots do not show any variation in the different conditions. Therefore Haider et al. were able to conclude that there is not a difference between the activated T-cells and the purified T-cells.
When gene expression was looked at for PBMC’s cultured for twenty four hours with the various conditions, there was more than 1,000 genes whose expression increased by 1.2 fold; which Haider et al. concluded to be statistically significant. In contrast, less then half as many genes were upregulated in the purified T-cells (Figure 2). This result shows that T-cells could be regulated by accessory cells because genes found to be upregulated in activated PBMC’s are not typically associated with T-cells and were myeloid cells. In psoriasis lesions myeloid cells are highly upregulated. These genes were separated by using heat maps where clusters of genes regulated by TNF or IFN in PBMC’s increased at significant levels (Haider et al.).
The next part of the study identified genes regulated by cytokine-neutralizing antibodies in activated PBMC cultures. Haider et al. strove to define the realm of genes which are regulated by cytokines by measuring the change in gene expression in PBMC’s in the presence of antibodies which block TNF or IFN. The antibodies bind to TNF or IFN produced after the activation of the T-cells in the activated PBMC’s and therefore inhibit the expression of the genes regulated by these cytokines. There are two types of antibodies produced; the first neutralize cytokines and the second is nonspecific. Again, Haider et al. used heat maps which showed that seven genes were significantly downregulated by 1.2 fold.
In the next part of the study, Haider et al. identified genes suppressed by blocking IFN in active PBMC’s. There has already been a great deal of research on IFN and genes whose expression is regulated by IFN. Haider et al looked at the regulation of these genes as well as other genes. The addition of the cytokine inhibiting antibodies left eighty percent of genes associated with IFN and TNF without expression change. Genes previously found to be cytokine regulated showed a reduction in expression of at least 1.5 fold when treated with cytokine inhibiting antibodies. Haider et al. did find blocks of genes shown to be downregulated by IFN or TNF not previously associated with cytokine inhibitors. Using the heat maps, shown below in Figure 2 clear blocks of genes are shown to be regulated by either anti-TNF or anti-IFN agents. The data shows nine genes exclusively suppressed by fontolizumab (anti-IFN) and seven genes were found to be downregulated by both fontolizumab and infliximab (anti-TNF). IFN mRNA was found to be the most suppressed gene.
Figure 2:
The above heat graph shows genes regulated by IFN or TNF. The green areas of the graph show genes whose change in expression is less than 1.5 fold (downregulated) and the red areas show genes whose expression change is greater than 1.5 fold (upregulated). The heat map looks at the various conditions of treatment with control or anti-TNF and anti-IFN agents. The blocks of green in the sections treated with fontolizumab (anti-IFN) and infliximab (anti-TNF) clearly show downregulation of genes that are not downregulated in the control group showing that they are regulated by the anti-TNF and anti-IFN therapies.
Haider et al. then identified genes suppressed by blocking TNF in activated PBMC cultures. Using the same method for the identification of IFN regulated genes, the group found a total of fifty genes which were significantly suppressed by 1.5 fold with the treatment of anti-TNF infliximab. Again IFN mRNA was the most suppressed gene, it was suppressed by 9.5 fold. The reduction if IFN mRNA reduces IFN protein synthesis in the PBMC’s treated with infliximab.
Haider et al. looked at a set of six genes which have been shown to be exclusively regulated by IFN and fifteen genes regulated by TNF which are inflammatory genes in leukocytes that are also upregulated in psoriasis lesions. The data shows that treatment with anti-TNF and anti-IFN agents leads to the gradual reduction in levels of IFN mRNA in skin lesions. It also shows that infliximab regulates many classical IFN response genes and that in a chronic inflammatory disease TNF may be more associated with inflammatory products of long term immunity than an immediate inflammatory response. This overall reduction of the inflammatory response from long term treatment of ant-IFN agents causes the direct suppression of IFN mRNA synthesis in T-cells (Haider et al.). This reduction of IFN mRNA could explain increased risk of bacterial infections and tuberculosis found in patients treated with cytokine inhibitors.
Researchers agree that prolonged treatment with anti-TNF therapies weaken the patient’s defenses for fighting disease. The patient then becomes vulnerable to microbial infections as well as tuberculosis. Other severe side effects of the anti-TNF treatment include increased risk of lymphoma and other cancers. The reactivation of hepatitis B has been known to occur, serious liver injury can occur, blood disorders and allergic reactions are also side effects associated with anti-TNF drugs (Medical Dictionary).
Kang et al. did research to study the body’s response to anti-TNF treatment. TNF-alpha, which is a type of TNF inhibited by anti-TNF therapies, is an important inflammatory cytokine that is synthesized by several cells and may play a role in the acute phase response in the inflammation reaction. The study involved seventy patients who were genotyped and given various treatments of anti-TNF drugs, the response to the drugs were then measured and compared.They measured the amount of TNF-alpha produced by each patient and the various genes affected by TNF-alpha. The allele with the strongest association with response to anti-TNF treatments was the -857T allele. In addition, they found that high production of TNF-alpha is associated with a lack of response to anti-TNF treatment.
The study by Smith et al.examines the problem of the systemic effects on gene expression of anti-TNF treatment. They engineered a specific anti-TNF therapy which secretes a modified anti-TNF single chain antibodies (TN3CK) locally rather than systemically. TN3CK was found to have TNF neutralizing effects that resulted in a significant decrease in the percentage of test animals with arthritis. Smith et al.’s analysis of the cell proliferation and cytokine secretion in response to the treatments show that the treatment is not affecting the cytokines production. The treatment only caused local changes to inhibit the TNF. The localization of TNF inhibition allows the body to have a normal immune response that is not possible with systemic TNF inhibition. It is important to note that this treatment will not be useful for those afflicted with systemic autoimmune diseases but will be very useful for those with localized autoimmune diseases. Localized treatment would stop the alteration of gene expression as a result of anti-TNF and anti-IFN therapies and be much less harmful to the patient while still allowing the patient to receive treatment for their autoimmune disease. Anti-TNF and anti-IFN treatments have allowed patients with autoimmune disease to lead relatively normal lives. However, there is still a great deal of research that can be done on the cytokine inhibiting therapies and their effects on gene expression and the body’s reaction to the therapies.
References:
Autoimmune Disorders. Lab Tests Online. American Association for Clinical Chemistry. Accessed 3 November 2007
Autoimmune Disease Fact Sheet. American Autoimmune Related Diseases Association. Autoimmune Related Diseases Association Inc. Accessed 5 November 2007 19e4c00ffe33c7cb6c43ed3aa>.
Haider, Asifa S. and Jules Cohen et al. “Insights into Gene Modulation by Therapeutic TNF and IFN Antibodies: TNF Regulates IFN Production by T Cells and TNF-Regulated Genes Linked to Psoriasis Transcriptome” Journal of Investigative Dermatology 11 October 2007
Kang et al. “The influence of a polymorphism at position –857 of the tumour necrosis factor gene on clinical response to etanercept therapy in rheumatoid arthritis” Rheumatology (2005) 44:4, 547-552. < 7ae7bd02895d27a06fe746c36bc84a92a>.
Krueger, JG. “The immunological basis for the treatment of psoriasis with new biological agents.” Journal of the AmericanAcademy of Dermatology (2002) 46:1, 1-23. 3 =11756941&dopt=Citation>.
Lackie, John. “PBMC.” The Dictionary of Cell and Molecular Biology Online 5 Accessed 1 November 2007
Medical Dictionary. Medicine Net Inc. Accessed 1 November 2007<
Smith, R., Tarner, H. et al. “Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis.” Gene Therapy (2003) 10, 1248–1257. Nature Publishing Group. >.