Cytokine Testing in Clinical Pain Practice

If you attended medical school more than twenty years ago, you may be unfamiliar with the significance of cytokines in modern medicine. As an example of current interest in the topic, there are almost five million hits on this subject on Google and 419,000 on Pubmed. Reviewing the type of work being reported, the nature of the first problem with cytokines is immediately evident as far as the clinician is concerned. The great majority of the work is pure research, complex and often relates to animal models. To say it is esoteric is an understatement; the human research frequently relates to end-stage diseases and tropical medicine. There are virtually no papers on the utility of cytokine measurement in traditional clinical medicine—the variety of medicine practiced in every hospital and medical clinic in America. This paper will suggest the use of cytokine testing as a screening tool—something like a sedimentation rate but better.

Nearly every subspecialty of medicine is represented in the work being done on cytokines. Certainly the most studies might be encountered in rheumatology, but they may be seen in the fields of immunology, neurology, headache and pain medicine, internal medicine, dentistry, dermatology, oncology, and many others.

About Cytokines

Cytokines are a type of signaling molecules and are important in the intercommunication between body cells.¹˒² They are most often discussed in the context of immune response, but are also significant factors in the genesis of autoimmune disorders. They may be proteins, peptides or glycoproteins, and can be produced by a diverse population of cells and affect a number of different target cells. They differ from hormones in that they are not produced by a single organ, nor are they likely to have a narrow target range.

There is a dizzying array of these substances discussed in the literature, with subsets being divided into six basic categories. These include interleukins (of which there are at least 18 varieties and are produced by leucocytes or macro-phages and act on other leucocytes), chemokines (which are important in attracting immunologically active cells to an area and of which there are at least 24), colony stimulating factors, interferons, tumor necrosis factor subtypes, and growth factors. Unfortunately, the sheer number of—and the variable terminology used to name them—increases the confusion of this important topic.

The simplest way to explain cytokines is to say that their measured presence suggests the human body is reacting to something; they reflect trouble, some kind of threat, injury or specific infection. Since cytokines are not specific in themselves, they don’t make diagnoses, but rather become evidence in favor of diagnoses in conjunction with other evidence. They are circumstancial evidence so a history, physical and other testing are still required.

Testing Cytokines

We have been employing a panel of twelve cytokines, performed by ARUP laboratory in Salt Lake City who do a multiplexed fluorescent microsphere immuno-assay (Luminex Corp).3 These cover many of the cytokines most commonly men-tioned in the literature when reviewing the topic. Many others could be tested individually and other panels at other labs could be substituted, but we elected to use one well-recognized and well-qualified laboratory. ARUP Laboratories, owned by the University of Utah, is a national clinical and anatomic pathology reference laboratory engaged in research and development with an extensive test menu of highly complex and unique medical tests.Even doing this, it required several months to educate our local laboratory facilities to handle the tests correctly. We have also asked our patients to use only one lab when doing these tests to avoid confusion, mistakes and frequent phone calls to our office. For example, the sample must arrive frozen at ARUP or the whole process starts over again.

Summary of Twelve Basic Cytokines (ADA interleukins or IL’s)4-6

A discussion of normal levels of cytokine in serum (as determined by ARUP Laboratories) is presented below and summarized in Table 1.

Tumor Necrosis Factor Alpha (TNF-alpha). The presence of TNF-alpha was first suggested in 1968. The agent discovered was termed lymphotoxin. In 1975, it was renamed as TNF-alpha. It has numerous actions on various organ systems and often in association with other cytokines. It functions as an acute phase responder in the immune system and may effect many other organ systems as well. It causes local inflammation by attracting neutrophils. Beyond the utility of tumor necrosis factor in the immune system, the substance may produce other problems. With acute high dose exposure of TNF-alpha, there may be shock and tissue damage, gastrointestinal necrosis, catabolic hormone release, vascular leakage syndrome, adult respiratory distress disorder and death. With chronic exposure there can be weight loss, anorexia, protein catabolism, lipid depletion and hepatosplenomegally. This substance has been correlated with numerous disease processes, most often autoimmune disorders such as rheumatoid arthritis, AIDS, ankylosing spondylitis, psoriasis, and Chrohn’s disease. One problem with TNF-alpha testing is that it may only appear in serum or spinal fluid for a very short period of time at the onset of an illness and immune response and, therefore, would be missed on later testing. Average serum level of TNF-alpha in normal subjects (as determined by ARUP Labs) is 1.3pg/ml

IL-1 and IL-1b. Produced by a variety of cells including NK and B cells. They cause growth and proliferation of immunological cells and may cause inflammation and may lead to fever. IL-1b is the one tested in the routine panel. An increased level of IL-1b has been seen in RA, sepsis, cachexia, leukemia, inflammatory bowel disease, AIDS, and transplant reactions. Higher levels have been associated with an increased risk of myocardial infarction (MI). ARUP lists IL-1b as normal at 0-36 pg/ml. In a recent study of 109 normal subjects, they found an average serum level of 13.5 pg/ml.

IL-2. Produced by thymus cells, IL-2 will cause activation and growth of immunological cells. It has been used as a cancer treatment. The ARUP average blood level is 1.6 pg/ml.

IL-2r. This is a reflection of the IL-2 receptor and is released into circulation under a variety of circumstances. The best known correlation is with organ transplant rejection, but there is also considerable interest in IL-2r associated with lymphoma, Hodgkins disease and leukemia. Otherwise, it may be noted in a great variety of autoimmune disorders, nephropathy and renal failure. The ARUP average level is 502 pg/ml.

IL-3. This is not a routine member of the cytokine panel. It is a cytokine produced by leukocytes and other cells in the body and increases the number of blood cells being made by the marrow. It can be used as a form of cancer therapy to boost the immune system in cancer therapy.

IL-4. This cytokine is produced by leukocytes and other body cells and causes B and T lymphocytes to increase in number and produce antibodies. IL-4 produced in the laboratory can be used as a form of cancer therapy. The ARUP average level in serum is 0.1.

IL-5. This cytokine is produced by helper 2 cells and mast cells. It induces the growth of B cells and increases immunoglobulin secretion. It activates eosinophil and is associated with several allergic disorders including allergic rhinitis and asthma. The ARUP average level is 5.3 pg/ml.

IL-6. This cytokine is both a pro-inflammatory and anti-inflammatory substance. It is secreted by t-c cells and macrophages. It plays a role in the response to tissue trauma or burns. It causes fever. The ARUP average level is 4.0. Increased serum levels may be found in cases of sepsis, autoimmune disorders, lymphomas, AIDS, alcoholic liver disease, tumor development, Alzheimer’s disease and transplant rejection.

IL-7. This interleukin is particularly important in the proliferation of immunological cells. It stimulates the growth of T and B lymphocytes. It can be used as therapy to increase the immune system’s response to cancer. It is not included in the routine twelve member panel.

IL-8. This interleukin is a chemokine produced by macrophages and other cell types. It is one of the major mediators of the inflammatory response. It has been associated with infection and autoimmune disorders such as psoriasis. We have observed it in the spinal fluid of many of our patients with new daily persistent headaches. The ARUP average serum level is 4.5 pg/ml.

IL-10. This cytokine, produced by monocytes and phagocytes, is also known as human cytokine synthesis inhibitory factor. It has multiple effects in immunoregulation and inflammation. It inhibits the synthesis of pro-inflammatory cytokines like IFN-g, IL-2, IL-3, TNF-alpha, and GM-CSF. It may also stimulate certain T cells, mast cells and B cells. The ARUP average serum level is 10.7 pg/ml.

IL-12. This interleukin is naturally produced by dendritic cells, macrophages and lymphocytes and is a T cell stimulating factor and increases the production of interferon gamma and TNF-alpha from T cells and natural killer cells. Its presence in spinal fluid has been associated with severe head trauma. It can be made in the laboratory to boost the immune system. The ARUP average serum level is 1.4 pg/ml.

IL-13. This is a cytokine created by many cells, but especially T helper cells, and is an important mediator of allergic inflammation and disease. It has often been associated with response to parasitic infection in humans. The ARUP average serum level is 0.8 pg/ml.

IFN-g. This is a cytokine that is member of the class of interferons. It is important in the body’s response to viral and bacterial infections. The ARUP average level is 0.2 pg/ml.

Recent Literature Relating Cytokines to a Variety of Disease States

The following snippets are a sampling of the enormous amount of literature in the cytokine area.

Articles on cytokines associated with a variety of infectious diseases are notable in the literature. One of the most common areas of research relates to cerebrospinal fluid levels of various cytokines associated with meningitis of various types. TB and malaria are both represented. AIDS is often associated with increased serum levels of cytokines, particularly TNF-alpha. Serum levels of cytokines may reflect a poor prognosis in sepsis. The presence of cytokines has been studied in numerous other specific types of infection.⁷⁻⁹

There have also been numerous studies on cytokines in cancer patients. Particular note has been made of the high levels of serum IL-2r in cases of lymphoma, Hodgkins disease and leukemia. IL-8 production has been shown to be associated with central nervous system tumors. Cytokine levels have been studied in numerous other varieties of cancer, including lung, prostate, ovarian and pancreatic.¹⁰˒¹¹

There have been multiple studies demonstrating the association of cytokines with headache— both migraine and new daily persistent headache. Serum cytokine levels are reported to be increased during migraine attacks. TNF-alpha levels have been noted to be increased in the spinal fluid of new daily persistent headache and chronic migraine patients.¹²˒¹³

Most notably, numerous studies on chronic fatigue syndrome have demonstrated increased levels of cytokines in spinal fluid.¹⁴

In the psychiatric realm, increased levels of cytokines have been noted in association with certain disease processes. Increased IL-6 in the cerebrospinal fluid of certain schizophrenic patients has been noted, as well as other abnormalities associated with depression.¹⁵˒¹⁶

Increased levels of tumor necrosis factor alpha have been found in the spinal fluid of autistic patients.¹⁷

Multiple studies have discussed the relationship between multiple varieties of autoimmune diseases and cytokines. IL-6 has been increased in the spinal fluid of lupus patients with central nervous system involvement, for example. Both tumor necrosis factor and IL-6 have been elevated in patients with psoriasis.¹⁸

Significant head trauma has been associated with increased levels of CSF IL-12.¹⁹

There have several reports of increased csf cytokines in association with Alzheimer’s disease and one claim of improvement in an AD patient with an anti-TNF-alpha agent.²⁰

We have also noted remarkable abnormalities in the serum cytokine panel in a single patient with neurofibromatosis (not reported before as far as we could determine).

There has been one report of increased serum tumor necrosis factor alpha in a series of narcolepsy patients.²¹

There have even been reports of increased serum cytokine levels associated with the alcohol hangover state.²²

Testing of cytokines has been performed in a variety of body fluids including serum, cerebrospinal fluid, saliva and synovial fluid.

The samplings above are just the tip of the iceberg as far as cytokine research is concerned. I have no doubt that a researcher in the field could spend a lifetime devoting himself to a single disease state. The majority of the papers in this field relate to small numbers of patients and, of course, cannot state whether the cytokines measured are causative or simply epi-phenomenon in the diseases in question.

Unfortunately, it cannot be stated with any certainly that the testing procedures in this field are accurate. It is been quite difficult for our group to determine the normal values of both serum and csf cytokines with so much variability reported in the literature. It was for this reason that we were thankful to receive the study by ARUP laboratory on their observation of normal serum levels in 109 subjects. Beyond our experience with our own patients, we have yet to be sure of normal csf cytokines.

In spite of all these doubts and concerns, however, the field of cytokine research presents great possibilities for the clinical practice of medicine.

Potential Value of Cytokine Testing in Clinical Medicine

As we have done the cytokine panel as a routine study we have found a number of useful applications for the testing. Since all the cytokines may play a role in multiple pathological conditions, the findings are not specific. They are however valuable in a number of ways that will be described below and summarized in Table 2.

1. As a test which increases your index of suspicion as to the presence of an underlying disorder (most often auto-immune in nature). In most cases, cytokines reflect the body’s reaction to an infection, tumor or injury. Therefore, they can reflect the presence of illness without giving specific data as to the nature of that illness—very much like a sedimentation rate or CRP. In a way, cytokine testing could be considered the new and improved sedimentation rate.

Case 1. Cindy, age 29. This patient is a severe migraine patient who is on abortive and preventative meds for treatment. She has no other illnesses nor any suggestion of autoimmune disorder by history. ANA, c-reactive protein and ESR are normal. Her serum cytokines are distinctly abnormal (however testing was not performed during a migraine). There is mild elevation of IL-4, 13 and interferon gamma. There is a moderate elevation of IL-2, 5, 10 and 12. There is a marked elevation in IL-1b. In her case, the significantly abnormal serum cytokines should ring some alarm bells that we are missing an underlying illness. I would be most concerned about autoimmune or neoplastic disease.

Case 2. Kathryn, age 38. This patient has had a long history of severe recurrent migraine. Beyond that, her symptoms have been nonspecific, including spinal pain and weight loss. ANA and other autoimmune studies have been negative. A recent cytokine panel was distinctly abnormal demonstrating an abnormal TNF-alpha of 20, mild elevation of interferon gamma and IL-13, and moderate elevation of IL-1b, IL-2, IL-6, 8, 10, and 12. As in the first case, our index of suspicion would be increased in her case because of the cytokine levels. Her body in reacting to something and thus far we don’t know what that factor may be.

2. As a test which correlates with a recognized disorder, most often autoimmune in nature.

Case 1. Cathy, age 46. This patient has a long history of Chrohn’s disease and is currently on treatment with Immuran. Two years ago, she had the abrupt onset of severe headaches and was diagnosed with new daily persistent headache. Neurological workup was negative including MRI of the brain, lumbar puncture (LP) and cerebrospinal fluid analysis. Autoimmune testing including the ANA panel was negative as was CRP and ESR. Recent serum cytokines were performed and demonstrated moderate elevation of IL-2r, IL-12, IL-4, IL-13 and IL-1b. CSF cytokines were not performed.

Case 2. Sherry, age 59. This patient has had a long history of Lupus with clinical difficulties involving rashes, chronic fatigue and arthralgias. Just recently her ANA was negative but the serum cytokines were significantly abnormal. Her tumor necrosis factor alpha was 23 (normal 1.3). Moderate elevations were noted in IL-1b, IL-2, IL-2r, IL-5, 10, 12 and 13.

What we found most notable in these two cases was the current negative routine autoimmune testing but with the significantly abnormal serum cytokines. It is certainly possible that other autoimmune testing, such as complement levels could have been abnormal at the time the serum cytokines were done, but these studies were not done in this project. It would seem a very reasonable future study to compare serum cytokines to the levels of other autoimmune testing.

3. As a test which supports the diagnosis of an autoimmune disorder in the absence of abnormality in the traditional studies (such as ANA, ESR, and CRP). These are patients not previously diagnosed as having an autoimmune disorder.

Case 1. Leslie, age 54. This patient had a history of Hodgkins disease at age 28 treated with RT and chemotherapy. She also had breast cancer at age 49 treated with chemotherapy. Following the breast cancer treatment, she developed a syndrome of generalized pain and arthralgias, diagnosed as possible fibromyalgia, along with a continuing problem with migraine. She was seen by rheumatology and treated with opiates for her significant chronic pain condition. Lab work of significance included negative ANA, thyroid, ESR, CRP, cbc and cortisol.

More recent cytokine testing in the context of her unchanging chronic pain disorder demonstrated significant abnormalities including mild elevations of IL-2r and IL-12 as well as marked elevation in IFNg, IL-4, IL-5, IL-13, IL-1b. TNF-alpha was normal. Diagnosis: post RT/chemotherapy autoimmune disorder with no indication of recurrent tumor at this point. We found the cytokines of particular note in her case because some of the clinicians in her case were not convinced of the organic nature of her illness.

4. As a test which raises particular concern about the presence of a tumor, often of lymphoid nature.

The cytokine IL-2r is the one most often associated with the presence of tumor, usually lymphoid in nature. We have observed three patients with a history of Hodgkin’s disease and lymphoma, all of whom were in remission but still had moderately increased IL-2r levels. More troubling than that, we have encountered a great number of severe headache patients with markedly elevated IL-2r levels without explanation. This included one 16-year-old male with new daily persistent headaches. Workup for cancer had been normal in a small group of these patients thus far, but our concerns about the possibility of occult neoplasm remain. Their physicians, on the whole, have not been interested in pursuing a major cancer evaluation without more data in hand. These doctors included oncologists as well as primary care doctors.

5. As a test in patients with a recognized infection.

Case 1. Kurt, age 59. Kurt has a long history of hepatitis C treated in the past with interferon. In association with that he has had a long history of severe intractable chronic daily headaches which were being treated with very high doses of opiates. His routine autoimmune workup has been negative but his cytokines have been remarkably abnormal.

While his serum cytokines only show one abnormality—a modest elevation of IL2r— his spinal fluid cytokines, however, are grossly abnormal with TNF-alpha of 21 (normal 1.3), IL-2, 4, 5, 6, 10 and 13 moderately elevated and IL-1b greater than 1000 (normal 13.5). We have seen this discrepancy between spinal fluid and serum levels of cytokines on multiple occasions. In most cases of new daily persistent headaches, we found increased cerebrospinal fluid IL-8 levels but not increased serum levels. In this case, we assumed there was evidence of active CNS disease based on the cytokine level in the spinal fluid and the blood brain barrier prevented the cytokines from moving into the serum. As far as we know, this observation regarding high CSF cytokines has never been reported before in hepatitis c.

6. As a test which may explain a set of clinical observations.

Tumor necrosis factor alpha is well known to produce a variety of symptoms and has been used as a treatment and given intravenously to patients. The presence of tumor necrosis factor alpha, in and of itself, may explain weight loss and loss of appetite or more severe life threatening issues such as shock.

7. As a test that may support the impression of a non-organic condition (when entirely normal) in conjunction with other studies.

8. As a test which may have value for other specific illnesses once more data is collected (i.e., psychiatric disease, autism, Alzeimer’s disease, narcolepsy).

Case 1. Linda, age 57. This patient has a history of neurofibromatosis. In the past several years she has developed a neurofibroma in the cervical region which the surgeons have elected to watch without surgery. A recent serum cytokine panel was performed and demonstrated mild elevation in IL-2r, IL-5 and IL-10. Moderate elevation was noted of IL-1b and IL-13. Review of the literature revealed no information on cytokine testing in neurofibromatosis. Discussion of the topic with physicians working in a university neurofibromatosis clinic was notable in that none of them had heard of this phenomenon.

Conclusions

In spite of the tremendous interest in cytokines in the literature, there is little practical information available for the practicing physician on the subject. Cytokine testing is currently readily available and panels of multiple cytokines can be done without difficulty.

It is quite evident that cytokine testing may be useful as a screening tool, akin to sedimentation rates and CRPs. In other cases, there may be specific value in testing as a follow up of autoimmune diseases or initial evidence in favor of that diagnosis. Certain test results may suggest the presence of lymphoid tumors or recurrence of these tumors (IL-2r). Finally, certain specific disorders such as narcolepsy, Alzeimers or autism may be associated with abnormalities in cytokine levels. In these cases, the presence of cytokines in increased amounts in the serum or spinal fluid may give us insight into the nature of these diseases and, beyond that, potential treatments not yet considered.

A colleague asked me if we really needed yet another blood test in the autoimmune panel. After all we have sedimentation rates, CRP, complement level, ANA and all the subcategories of antibodies for specific autoimmune disorders already at our fingertips. My response is that screening tests are always valuable in medicine, even when they are not absolutely specific. Using screening tools in medicine is like playing “Twenty Questions” (what we used to call “animal, vegetable or mineral”). The correct answer is best discovered by offering broad questions initially to rule out whole categories and finishing with more specific questions dealing with smaller populations. The good feature of cytokine testing is that it shows the patient is reacting to something; the weakness is the lack of specificity.