Testing systemic cytokines

Arthrofibrosis occurs when myofibroblasts (the cells that make fibrosis) continue to create scar tissue, adhesions and contractions after they should have disappeared. Myofibroblast activation occurs as a result of inflammation. But the biological processes that make inflammation and fibrosis are complex, and every individual has their own unique makeup that affects their recovery after injury or surgery. Most people will have a short term spike in inflammation after injury and surgery and recover, but for some of us, the result is chronic (long term) inflammation.

It’s important to understand what the main fibrosis drivers are for each individual

Even systemic (whole body) low-grade inflammation that is not symptomatic can keep myofibroblasts activated, so we need to know if inflammation is present. Unfortunately, the traditional markers used for detecting systemic inflammation, serum CRP and ESR, are not specific or very sensitive, so a lot of people with systemic inflammation are not diagnosed and treated appropriately. Even in autoimmune conditions such as spondyloarthritis (this covers a range of conditions including ankylosing spondylitis) around 60% of patients do not have elevated CRP levels, and measuring CRP is not useful for these people [1]. Yet, CRP continues to be the marker that many physicians base their diagnosis of spondyloarthritis on, together with joint swelling [2]. It’s interesting that a number of IAA members have the HLA-B27 marker associated with spondyloarthritis. It’s a common disease that can affect tendons and joints, and is very difficult to diagnose in the early stages [1].

Figure: example of a cytokine panel result for an arthrofibrosis patient

There are two main causes of chronic inflammation: an inappropriate immune reaction against self (your own cells), or prolonged exposure to an inflammatory stimulus (environmental causes) [3]. In the first group, biology (genetic makeup) is the most important driver that activates chronic inflammation. Let’s call these the “immune-activated” group, which covers a variety of immune system irregularities. My own test results indicate that my body is immune-activated, but like many other people in this group I was fit and healthy, and my inflammatory status was not apparent until I got arthrofibrosis.

For other people, arthrofibrosis may have been primarily caused by prolonged exposure to an inflammatory stimulus (environmental causes), such as aggressive physiotherapy and high activity levels after surgery, or a misplaced ACL or prosthesis. This group doesn’t have systemic chronic inflammation, so for these people their biology likely plays a less significant role in their arthrofibrosis pathology. We’ll call these people the “immune-regulated” group. With appropriate care these people may recover from injury or surgery to a greater extent (compared to immune-activated people), even without the help of specialised medications.

So, it’s important to understand what the main fibrosis drivers are for each individual, as this affects the medications they need. But how do we “sort the sheep from the goats” – they look the same. That is, how do we know who belongs to which group, since on the surface there is often no way to tell.

We have access to more sensitive and specific tests, called cytokine test. These can be very useful for indicating which treatments will be the most effective.

Luckily, science is keeping up, and now we have access to more sensitive and specific tests, called cytokine tests. Cytokines are compounds the body makes for protection and healing, but they cause pathology when their production or signalling becomes dysregulated. A blood test to determine the levels of systemic cytokines is very useful for understanding an individual’s inflammatory status. An abnormally elevated level of one or more systemic cytokines suggests that the individual is in the immune-activated group. The classic inflammatory cytokines are TNF-α, IL-1 and IL-6, however there are quite a lot of them. Some cytokines are occasionally referred to as anti-inflammatory, but this label is misleading, because their actions are often context-dependant and in chronic inflammation they can become pro-inflammatory. Some of these, including TGF-β, IL-4 and IL-13 strongly stimulate fibrosis [4], so elevations in these are not a welcome result.

In addition to “sorting the sheep from the goats” (understanding if you’re immune-activated), a cytokine panel test can be very useful for indicating which treatments will be the most effective. There are many pathways that can become dysregulated3 and these may arise from innate or adaptive immunity, and be a Th1 or Th2 response. If certain serum cytokines are elevated, they can sometimes be specifically targeted with modern medications that are more effective at reducing inflammation. These medications may also have fewer side-effects that older medications, like corticosteroids, have. A good example of cytokine targeting is the TNF-α antibodies. TNF-α is a key inflammatory cytokine, and a number of IAA members have abnormally elevated TNF-α and would likely benefit from a TNF-α antibody.

The results of a systemic cytokine panel test can be used by a rheumatologist to prescribe the most appropriate medication, so the treatment is tailored to the individual. An injected biologic called golimumab (Simponi) binds to both soluble and membrane forms of TNF-α and blocks the action of this cytokine. There are a number of similar TNF-α antibody medications available that have slightly different antibody structures and effects, and if you have elevated TNF-α you may need to experiment to find one that works well for you.

Metformin can help by reducing TGF-β levels and inflammation, and is one of the world’s safest medications. People who only have elevated IL-1β might have inflammasome (threat recognition complexes) mutations and can be effectively treated with biologicals such as anakinra that block this cytokine [5]. Those with a suite of elevated inflammatory cytokines might benefit from a medication that blocks a common signalling pathway, such as a JAK inhibitor (eg Rinvoq), which has broader immune suppressant effects. Another recently developed therapeutic option are the biologics that target integrins [6]. Integrins are cell adhesion molecules that myofibroblasts need to stay activated. 

There are other medications that might be prescribed by a rheumatologist based on blood test results. Please remember that every treatment has potential side-effects that you should be aware of. Differences in biology mean that some people experience side-effects when others don’t, or have better response to a particular medication. The risk of negative side-effects needs to be balanced against the risks of not treating the inflammation, since it’s well known that chronic inflammation contributes to many diseases [3]. 

In addition to cytokine tests, blood tests for thyroid function, insulin levels, autoimmune conditions (especially HLA-B27 and rheumatoid factor), general chemistry, infection and complete blood count are recommended for people with arthrofibrosis. A rheumatologist is best placed to order and interpret these tests, but some of them can be ordered by a general practitioner. Cytokine panel testing is relatively new, and some physicians prefer to rely on old methods that are not very useful for people with arthrofibrosis. So, you might have to be proactive and ask your doctor to prescribe a cytokine panel test, and the cost probably won’t be covered by health insurance. You will probably need to call the laboratory that performs the testing to order a kit (see below). Often, the lab will send you forms to complete along with express postage return for the sample, and some companies will also send a sample vial. Blood is then drawn in the usual way by a doctor or nurse into a sample vial. With express courier delivery the sample doesn’t need to be frozen.

If you’re given the option, prioritise TNF-α, IL-1, IL-17 and IL-6 as well as TGF-β, CTGF, IL-4, IL-13, IFN- γ and GM-CSF. The more tests you’re able to add, the more complete the picture is. Levels of some cytokine receptors (molecules that cytokines dock with and signal through) are sometimes added in order to understand a person’s biology, e.g. IL-2R, but this varies depending on the test laboratory. In depth genetic testing (other than the standard tests for autoimmune conditions) are interesting but expensive and they’re quite new so the results are less well understood.

I’ll address a couple of questions I’ve had about cytokine testing

• What about testing for inflammation in the joint with arthrofibrosis? Synovial fluid can be extracted by needle and tested for cytokines, but I feel that this is less useful than testing the levels of systemic cytokines, because a joint with painful arthrofibrosis is very likely to be inflamed. So, the information is unlikely to tell you anything you didn’t already know, assuming that infection has been ruled out. In addition, sampling synovial fluid is an invasive procedure that can stimulate more inflammation and bleeding. In contrast, testing the levels of systemic cytokines will tell you if you have systemic inflammation that is driving the joint pathology, and the results can point to a specific treatment. Some conditions are well known to cause joint pathology, including spondyloarthritis, and this condition is very difficult to diagnose in the early stages.
  

• The levels of systemic cytokines fluctuate, are test results reliable? Yes, the levels of systemic cytokines are not constant, they fluctuate depending on your recent activities, time of day, hormones etc. However, there is a known range of normal levels, and abnormally high levels of a cytokine are abnormal and suggest that attention is needed. Abnormal results can be verified with another test if necessary.
  

• Some cytokines are stored in tissues, is measuring their levels in blood reliable? Yes, TGF-β is one of these and is stored in tissues. It’s also the key cytokine in fibrosis pathology. Low TGF-β levels don't necessarily mean there is no problem with TGF-β, because the signalling of this cytokine might actually be abnormally high. However, if you saw abnormally high levels of systemic TGF-β then that would be of interest. To clarify, you’re more likely to get a false negative result than a false positive result.

Please understand that putting people into neat categories is a useful tool for guiding treatment, however, like any tool it can be misunderstood. Biology is a complex interplay of processes that follow a continuum without distinct cut-offs. So, biology doesn’t fit into neat boxes and these categories of immune-activated and immune-regulated shouldn’t be taken too literally. There will be cross-over between the groups. For example, environmental factors will also impact people in the immune-activated group and there may be some genetic predispositions in the immune-regulated people.

Companies that offer cytokine panel tests include ARUP Laboratories, the Mayo Institute and Quest Diagnostics in the USA, IMD Labor Berlin in Germany and NutriPATH in Australia. Abnormal test results suggests that a rheumatologist should be consulted.

References

1. Navarro-Compan, V., Sepriano, A., El-Zorkany, B. & van der Heijde, D. Axial spondyloarthritis. Ann Rheum Dis 80, 1511-1521, doi:10.1136/annrheumdis-2021-221035 (2021).

2. Bolt, J. W. et al. Treatment decisions in axial spondyloarthritis daily clinical practice are more than treat-to-target. Rheumatology (Oxford), doi:10.1093/rheumatology/kead155 (2023).

3. Germolec, D. R., Shipkowski, K. A., Frawley, R. P. & Evans, E. in Immunotoxicity Testing: Methods and Protocols (eds Jamie C. DeWitt, Cheryl E. Rockwell, & Christal C. Bowman) 57-79 (Springer New York, 2018).

4. Di Maggio, F. M. et al. Portrait of inflammatory response to ionizing radiation treatment. J Inflamm (Lond) 12, 14, doi:10.1186/s12950-015-0058-3 (2015).

5. Spel, L. & Martinon, F. Inflammasomes contributing to inflammation in arthritis. Immunol Rev 294, 48-62, doi:10.1111/imr.12839 (2020).

6. Zhang, J. et al. Discovery of a new class of integrin antibodies for fibrosis. Sci Rep 11, 2118, doi:10.1038/s41598-021-81253-0 (2021).