Clinical application of the complement system: A historical perspective

HOW WAS THIS CASCADE OF PROTEINS DISCOVERED?

Looking at this complex system of proteins, it’s easy to wonder how someone could have possibly discovered it, especially in the late 1800s, at the dawn of our understanding of the immune system. I’ll provide a little background: scientists were trying to understand how the immune system killed organisms via innate (Dr. Elie Metchnikoff describing phagocytosis) and humoral immunity. To better understand humoral immunity, Dr. Richard Pfeiffer injected cholera bacteria into the abdominal cavity of guinea pigs previously immunized to cholera, then collected serum from the animals and visualized the bacteria losing motility and eventually dying.³ This protection against cholera was unique to the cholera organism—animals immune to cholera would die when inoculated with other bacterial organisms.

Interestingly, serum from immunized guinea pigs added to cultured cholera ex vivo did not kill the cholera bacteria.³ Pfeiffer hypothesized that cells within the abdominal cavity of the guinea pigs, along with the immunized serum, were necessary to cause the bacteriolysis.

Around this time, Belgian scientist Dr. Jules Bordet was working at the Pasteur Institute, studying Pfeiffer’s work. Bordet realized that immunized serum can kill cholera bacteria ex vivo, as long as the sample was fresh and hadn’t been exposed to significant temperature fluctuation. The serum would lose its bacteriolytic ability if exposed to temperatures above 55°C (131°F). This was different from antibodies, which were known to be heat stable. Interestingly, if the serum was heated above 55°C and serum from a nonimmunized guinea pig was added to it, the original antiserum would regain its bacteriolytic properties. Bordet concluded that bacteriolysis was dependent on organism-specific antibodies and a heat-sensitive component not specific to the organism that complements the antibody killing of bacteria.⁴ He described what would eventually be called complement.

It would take decades of work to unravel the full spectrum of the complexity of the cascade. Much to the consternation of medical students, the individual components of the cascade were named in the order of discovery, not their place within the enzymatic cascade.

BRINGING COMPLEMENT TO DIAGNOSTICS: COMPLEMENT FIXATION TESTING

In 1901, Dr. Bordet developed a multistep laboratory test to check serum for antibodies to certain infections, taking advantage of the heat lability of complement and heat stability of antibodies. The complement fixation test was developed decades before the extent of the cascade was known. Here is an overview of how the test is conducted and its results interpreted:⁵

First, draw the patient’s blood, centrifuge it to separate the cells from serum, then heat the serum to 56°C (132.8°F), denaturing any complement in the serum but still preserving antibodies.

Then, add a standardized amount of complement (usually from a guinea pig) along with the antigen of interest to the heated serum and incubate the combination for multiple hours at 4°C (39.2°F). If antibodies are present in the serum, the antibodies will bind to the antigen, forming an antibody-antigen complex, triggering complement activation (and complement consumption).

Finally, add antibody-coated sheep red blood cells to the serum.

• If antibodies were initially present against the antigen, then complement will have been consumed and will not be available to lyse the antibody-coated red blood cells and the red blood cells will remain whole; this is a positive test.

• If antibodies were not initially present, complement will still be present in the serum and will be activated by the antibody-coated sheep red blood cells and cause hemolysis; this is a negative test.

It’s impressive that scientists elucidated this immunologic phenomenon for diagnostics in 1901, as it’s hard to wrap your mind around it in 2026.

Complement fixation allowed for a giant leap in diagnosing certain infections. Dr. August von Wasserman used the complement fixation test in 1906 to diagnose syphilis. Up to this point, syphilis was diagnosed based on pattern recognition of signs and symptoms, and latent cases were often missed. An initial barrier to using complement fixation to test for syphilis was that Treponema pallidum couldn’t be cultured, so it was difficult to obtain the bacteria to use as an antigen in the test. Wasserman got around this by harvesting tissue extracts from infected chimpanzees or the livers of human fetuses who died of syphilis. Once the Wasserman test was established, asymptomatic persons were tested in various settings, including prenatal clinics, military recruitment centers, and hospitals, revealing the true prevalence of syphilis as much higher than previously understood, over 10% in some populations.⁶

As syphilis testing became more widespread, multiple false positives were found in patients with other chronic infections, including tuberculosis and leprosy. A major surprise occurred in 1907 when it was found that healthy tissue from animals, particularly the myocardium of guinea pigs, could be used as the antigen in the Wasserman test. Over time it was discovered that chronic infections, including syphilis, cause tissue damage, releasing phospholipids from the tissue. The Wasserman reaction was detecting antibodies to the tissue damage, not antibodies to T pallidum, which also explained its positive rates in other chronic infections. Despite the lack of specificity, the Wasserman test was found to be a useful screening test for a disease that previously relied on symptoms and pattern recognition for diagnosis.

The clinical manifestations of syphilis are broad and heterogenous. In 1909, Wasserman testing was performed on a group of women who had multiple spontaneous abortions. It was thought at the time that the abortions were caused by syphilis, and this thinking was confirmed (falsely) when many of the woman tested positive. Utilizing animal myocardium allowed for the widespread testing of syphilis, but it wasn’t until the 1940s that the antigen cardiolipin was described.⁵ It would be decades later that anticardiolipin antibodies were linked to thrombosis and recurrent miscarriages.

Taking advantage of the complement system to diagnose a disease that previously relied on active signs and symptoms for diagnosis revolutionized our understanding of syphilis. The complement fixation test was developed without an understanding of the extent of the cascade or its function within the immune system. Over the next decades, the array of proteins within the complement cascade was elucidated, and testing for complement function, including testing for individual components, became possible, allowing for an understanding of complement’s role in disease.

COMPLEMENT’S ROLE IN DISEASE

It took many decades for the biological role of complement to be defined and its role in the pathophysiology of disease explained. In 1951, Vaughan and colleagues⁷ at Harvard published an article evaluating complement activity in 4 patients with active systemic lupus erythematosus (SLE), all of whom had low complement levels due to increased complement activity. The patients were treated with adrenocorticotropic hormone (glucocorticoid) therapy and experienced a clinical response along with normalization of complement levels.

In 1953, a report from the Mayo Clinic confirmed these findings by evaluating 27 patients with lupus compared with 60 controls. This study confirmed that patients with lupus had decreased measurable complement, and for the first time found a correlation between decreased complement and increased albuminuria, indicating kidney involvement.⁸ Testing for C3 and C4 in patients with SLE is now a standard method to help determine disease activity. We now know that the classical cascade is triggered in a number of immune complex–mediated diseases such as SLE, cryoglobulinemic vasculitis, urticarial vasculitis, and serum sickness.

Not only is complement important in the pathophysiology of multiple autoimmune diseases, but also a deficiency of components of the cascade can increase risk of infection with certain organisms. In 1974 at the University of Rochester, an 18-year-old woman presented with fevers and joint pain. She was eventually diagnosed with disseminated gonococcal infection, but because SLE was on the differential, complement levels were checked and were found to be low. Initially, the low complement activity was thought to be secondary to immune complex formation and complement activation due to the infection, but additional testing demonstrated a deficiency of C6, which is the first component of the membrane attack complex critical for bacteriolysis.⁹

Two years later, a 6-year-old was hospitalized with Neisseria meningitis for the third time. The young boy was also found to be deficient in C6, accounting for his repeated cases of the disease.¹⁰ These cases led to our understanding of the importance of the terminal complement components, mainly those leading to formation of the membrane attack complex, in fighting encapsulated organisms like Neisseria meningitidis and Neisseria gonorrhoeae.

OUR CURRENT UNDERSTANDING OF COMPLEMENT

The complement cascade is an intricate assortment of proteins cleaving and combining with one another to form difficult-to-memorize complexes. Well before the full extent of the cascade was appreciated, researchers took advantage of complement function in the laboratory to test for previously difficult-to-diagnose diseases such as syphilis. As laboratory techniques improved, we were able to measure complement activity, eventually measuring individual components of the cascade, allowing activity of complement to be recognized in certain diseases like SLE. Low levels of terminal complement were found to be associated with recurrent infections with encapsulated bacterial organisms. Measuring complement gave us a better appreciation of its role in disease, both in overactivity and deficiency. Now, our understanding of complement has evolved to therapeutic targeting of components of the cascade in a wide range of diseases, making it necessary for us to dust off the immunology book sitting on the shelf and figure out just what we’re shooting at.

DISCLOSURES

Dr. Brown reported consulting for Amgen and Sanofiand teaching and speaking for Amgen, Chemocentryx, and Sanofi.