Immune stimulation and elevated polyclonal free light chains

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21.1. Introduction

Figure 21.1 Serum FLCs in 25 patients with polyclonal hypergammaglobulinaemia (blue squares), compared with normal individuals (red crosses). (Courtesy of JA Katzmann).

Figure 21.2 Serum FLCs in different diseases compared with blood donors. Bars show range and mean concentrations of κ (red) and λ (blue). Juv chr arthritis: juvenile chronic arthritis. Juv FMS: juvenile fibromyalgia syndrome. Patient Nos. in brackets. (Courtesy of U Hoffmann).

Diseases associated with generalised increased B-cell activation frequently have high concentrations of polyclonal immunoglobulins and coexisting high polyclonal sFLCs (Chapter 29). This relationship was demonstrated in 25 patients studied at the Mayo Clinic ^[1]. In some patients, concentrations of both FLCs were highly elevated, although κ/λ ratios were always within the normal range (Figure 21.1). Total immunoglobulin concentrations were as high as 54g/L while maximum FLC concentrations were 273mg/L for κ and 307mg/L for λ. The correlation between immunoglobulin and sFLC levels was modest. This may have been due to impaired renal function elevating the FLC concentrations in some patients. However, data on GFR were not available.

One interesting aspect of elevated polyclonal FLCs is whether they are bioactive molecules in inflammatory disease processes. This has been mooted for some time but the availablity of a simple serum FLC assay has re-awakened this area of research.

21.2. Rheumatic diseases

Many rheumatic diseases feature polyclonal B-cell activation, high concentrations of autoimmune antibodies and polyclonal elevations of serum immunoglobulins. Excess polyclonal FLCs have been detected in the urine of these patients and indeed, their measurement may be useful for assessing disease activity. Presumably, serum analysis of FLCs in such patients would be more reliable. This is particularly applicable to patients with SLE, many of whom have high levels of urine polyclonal FLCs ^[2][3][4]. Since these patients frequently have renal impairment, sFLC concentrations may be highly elevated.

Hoffman et al. ^[5], investigated the relationship between sFLCs and other markers of disease activity in patients with rheumatic diseases. Figure 21.2 shows the concentrations of sFLCs in the different disease groups. Patients with intercurrent illnesses were excluded from analysis to ensure that the changes were due exclusively to the disease under study.

High FLC concentrations were found in rheumatoid arthritis, SLE, Sjögren's syndrome, vasculitis and systemic sclerosis compared with control groups of 28 patients with fibromyalgia and 19 blood donors (p<0.05). Furthermore, sFLC concentrations were more frequently elevated than intact immunoglobulins. In all individuals, κ/λ ratios were normal, indicating polyclonal synthesis. It was also found that sFLCs were more frequently elevated than C-reactive protein in patients with SLE, Sjögren's syndrome and systemic sclerosis. However, the numbers of patients in some groups were insufficient for statistical analysis. As might be expected, there was a positive correlation between concentrations of sFLCs and creatinine in all patient groups.

Figure 21.3 Serum FLC in SLE (A) with and (B) without renal involvement. The numbers of individuals studied are shown in brackets. Bars show range and mean concentrations of κ (red) and λ (blue). (Courtesy of U Hoffmann).

Figure 21.4 Serum κ FLC concentrations (and mean values) in patients with Primary Sjögren’s Syndrome (pSS), rheumatoid arthritis (RA) and healthy controls. NR: Normal range. (Reproduced with permission from Ann Rheum Dis and J-E Gottenberg^[6]).

Figure 21.5 Serum FLCs in relation to the presence of SSA and SSB antibodies in patients with Sjögren’s Syndrome. (Courtesy of X Mariette).

Systemic Lupus Erythematosus (SLE)

Hoffmann et al. ^[5], studied 45 patients with SLE and showed that sFLCs were elevated approximately 3-fold (Figure 21.2). Predictably, FLC concentrations were higher in SLE patients who had renal involvement compared with those having normal renal function (Figure 21.3).

Clinical scores of SLE correlated with sFLC levels, particularly when the disease was active. In a subsequent prospective study, the clinical scores (ECLAM) in 8 patients were compared with a variety of laboratory parameters ^[7]. sFLC concentrations showed a strong correlation with disease activity that was not observed for C-reactive protein or ESR.

Primary Sjögren’s Syndrome (pSS)

Gottenberg et al.^[6], studied 139 patients with pSS. 22% had raised sFLC, and mean levels were significantly higher than controls: p<0.001 (Figure 21.4), while κ/λ FLC ratios were normal in all but one patient. sFLC concentrations were significantly correlated with IgG (p<0.001), rheumatoid factor (p<0.005), β2-microglobulin (p<0.001) and B-cell activating factor (p<0.01).

Mean sFLCs were higher in patients with autoantibodies, particularly when both anti-SSA and anti-SSB were co-occurring (Figure 21.5). Also, patients with extra-glandular involvement had higher levels than those with only glandular involvement. Interestingly, 15 patients had monoclonal FLCs, a much higher proportion than might be expected by chance. These results indicate that extra-glandular involvement in pSS is associated with intense stimulation of B-cells.

Some of these patients progress to non-Hodgkin lymphomas, [particularly MALToma with an Odds ratio of 12.9 ^[8]] but currently no biological marker is available to evaluate the individual risk for lymphoma. However, the above results show that abnormal κ/λ ratios are associated with loss of control over the proportion of heavy and light chains synthesised. [Also, it was recently shown to be a relevant clinical marker of malignant evolution in B-cell CLL (Chapter 18) and MGUS (Chapter 19). Among 5 patients with pSS without MGUS who had abnormal κ/λ ratios, one had purpura and two had decreased C4 levels, both of which are risk factors for lymphoma.

No clonal B-cell populations could be detected in the blood of these patients, which suggests that an abnormal κ/λ ratio could be a more sensitive marker of clonality, possibly restricted initially to the site of autoimmunity. Hence, Gottenberg et al. ^[6], suggested that the predictive value of abnormal κ/λ ratios regarding the occurrence of lymphoma should be investigated in a longitudinal study of this disease.

Rheumatoid Arthritis (RA)

Gottenberg et al. ^[6], studied 50 patients with RA. 36% had raised sFLCs with mean values significantly higher than controls: p<0.001 (Figure 21.4), while sFLC κ/λ ratios were normal in all but 3 patients. sFLC concentrations were significantly correlated with IgG (p<0.04), C-reactive protein (p<0.04), and rheumatoid factor (for κ only: p<0.03), but not with anti-CCP. Significant correlations were observed between disease activity assessed by the Disease Activity Score 28 (DAS28) and both κ (p=0.0004 - Figure 21.6) and λ (p=0.05 - data not shown) concentrations. This supports the functional relationship between B-cells and disease activity. Interestingly, no correlation was observed between DAS28 and IgG, another marker of B-cell activation, but which has amuch longer half-life (20–25 days) than FLCs (2–6 h). The faster turnover of sFLCs might account for their observed disease activity correlation, and suggests that they might be a good early surrogate marker for responses to treatments. Further studies linking FLC concentrations to the use of drugs such as Rituximab that deplete B-cell numbers, and the development of clonal disease are underway.

21.3. Diabetes mellitus

Figure 21.6 Correlation between serum κ FLC concentrations and Disease Activity Score 28 (DAS28) in patients with rheumatoid arthritis. (Reproduced with permission from Ann Rheum Dis and J-E Gottenberg^[6]).

Figure 21.7 Serum FLC concentrations in 745 patients with early diabetes mellitus (circles) compared with the normal population (crosses) and showing patients with MGUS (diamonds) (1.9%). (Courtesy of C Hutchison).

Two early studies identified a relationship between urine FLC concentrations and rapidly progressive diabetes mellitus. Thus, 20 years ago, it was noted that patients with proliferative retinopathy had both higher urine κ FLC excretion (λ FLC assays were not available) than those without retinopathy and higher than in patients with proteinuria from other causes ^[9]. There was an associated elevated κ FLC/albumin excretion ratio. Subsequently, the same authors suggested that elevated FLC/albumin excretion ratios were an early indication of diabetic nephropathy and they directly implicated a renal cause of the FLC leakage rather than excess production ^[10]. This was supported by their finding of normal serum FLC concentrations. However, in the absence of sensitive serum assays this interpretation was, perhaps, premature.

A recent study by our group analysed FLC levels in both serum and urine of Type 2 diabetic patients to determine if they were an early marker of diabetic kidney disease ^[11]. It was clear that diabetic patients had significantly raised serum and urine concentrations of polyclonal sFLCs (Figure 21.7) before overt renal impairment developed (P<0.001). κ concentrations were higher than λ concentrations and 1.9% of patients had MGUS (confirmed by IFE). There was a good correlation between sFLC concentrations and various markers of GFR including serum creatinine, cystatin-C [κ; R=0.55 (P<0.01) λ; R=0.56 (P<0.01)], and estimated GFR (Figure 21.8 shown for κ only). South-Asian diabetic patients had higher sFLCs than Caucasian diabetic patients and this was independent of renal function suggesting more underlying inflammation.

Urinary FLC concentrations were raised in diabetic patients (P<0.001). 68% of patients with normal urinary albumin/creatinine ratios (ACRs) had abnormal urinary FLC/creatinine ratios. Urine FLC concentrations correlated with urinary ACR: κ, R=0.32, P<0.01 and λ, R=0.25, P<0.01 respectively. However, some patients had normal GFR (by MDRD) with high concentrations of sFLCs indicating increased production. This is suggestive of generalised inflammation/vasculopathy. Perhaps retinopathy and nephropathy are the most readily observed clinical signs of a generalised inflammatory process that is apparent from raised FLC production.

Since polyclonal FLCs are potentially nephrotoxic, increased concentrations may contribute to progressive nephropathy. It has also been suggested that monoclonal FLCs may play a role in some patients’ renal diseases ^[12]. Indeed, mesangial monoclonal FLC deposits observed in renal biopsies of patients with renal impairment are sometimes similar in appearance to those found in diabetic glomerulosclerosis ^[13]. Furthermore, FLC MGUS is observed in patients with renal impairment (Chapter 20), so it may be an additional risk factor for progressive nephropathy.

Thus, Type 2 diabetic patients have significantly raised concentrations of serum and urinary polyclonal FLCs before overt renal disease occurs. Possibly, measurement of polyclonal FLCs could provide a useful tool in early diagnosis of diabetic kidney disease.

21.4. Infectious diseases with elevated polyclonal serum free light chains

Figure 21.8 κ sFLCs in 745 patients with early Type 2 diabetes correlated with the estimated glomerular filtration rate - MDRD (p<0.001). (Courtesy of C Hutchison).

Figure 21.9 Serum FLCs in patients with pneumonia compared with other diseases. The slight increase in κ (black) compared with λ concentrations (blue) may be related to renal impairment. (Reproduced with permission from Zeitschrift für Rheumatologie and U Hoffman^[5]).

There was one early report by Solling ^[14], and a more recent study of patients with acute pneumonia (Figure 21.9) by Hoffman et al. ^[5] As expected, both publications showed that polyclonal sFLCs were elevated. In the latter study, the median κ/λ ratio was higher than in other disease groups suggesting modest impairment of renal function (Chapter 20).

One potent cause of elevated polyclonal immunoglobulins and sFLCs is chronic viral infection. Terrier et al. ^[15], studied 59 patients with chronic hepatitis C virus infections (HCV) and mixed cryoglobulinaemia (MC) at different stages of evolution to non- Hodgkin lymphoma (NHL). The MC comprised type II cryoglobulins with immune complexes of monoclonal IgM directed against polyclonal IgG. 17 patients had no MC, 7 had asymptomatic MC, and 35 had MC vasculitis, 9 of whom had B-cell NHL.

The results showed elevated sFLCs in nearly 50% of patients. Furthermore, mean polyclonal sFLC concentrations and the frequency of abnormal sFLC κ/λ ratios progressively increased with worsening disease category (Figure 21.10) (p<0.001 and p=0.002 respectively), increasing cryoglobulin concentrations (P<0.0001 and P=0.0016 respectively) and the severity of the B-cell disorder (P=0.045 and P=0.0012, respectively). Among patients with an abnormal sFLC ratio at baseline, FLC ratios correlated with the virological response to HCV treatment (Figure 21.11). The authors concluded that in HCV-infected patients, abnormal sFLC ratios were very interesting markers, and were consistently associated with the presence of MC vasculitis and/or B cell NHL. After anti-viral therapy, the serum FLC ratio could be used as a surrogate marker for the control of the HCV-related lymphoproliferation.

21.5. Free light chains as bioactive molecules in inflammatory diseases

Figure 21.10 In virus C hepatitis, abnormal sFLC ratios are progressively more frequent with worsening disease category. (MC: mixed cryoglobulinaemia. B-NHL: B cell NHL). (Reproduced with permission from Ann Rheum Dis and B Terrier ^[15]).

Figure 21.11 Abnormal sFLCs ratios in virus hepatitis C infections may normalise with successful anti-viral treatment. (EOF: end of follow-up). (Reproduced with permission from Ann Rheum Dis and B Terrier ^[15]).

Since FLCs are part of the binding site of intact immunoglobulin molecules they have bioactivity. This has been observed for both polyclonal and monoclonal FLCs and has recently been reviewed (Figure 21.12) ^[16][17].

For instance, biological activities of FLCs have been shown in patients with immediate hypersensitivity-like responses and contact sensitivity dermatitis. Since FLCs can activate mast cells (which contain a range of biologically active molecules), their potential for causing or contributing to inflammatory and other diseases such as asthma is high. Possibly, FLCs are one of the components in the active inflammatory processes that are apparent in chronic renal failure. Furthermore, different monoclonal FLCs have been shown to bind specific target molecules that can stimulate antiangiogenic activity or have proteolytic potential. The complementarity determining regions of FLCs have sufficient variability and flexibility that they could mimic almost any biological molecule.

In this context, removal of FLCs using “high cut-off” dialysers may be helpful in reducing inflammation in chronic kidney disease (Chapter 13). There has also been a suggestion that their inflammatory actions might be usefully blocked by novel FLC binding peptides ^[17]. To help resolve these issues, studies on purified polyclonal FLCs from patients with different diseases are required.

Figure 21.12 Biological actions of sFLCs include enzymatic acitivity, binding to tissue substrates and mast cells. (Reproduced with permission from Trends Pharmacol Sci and M Thio ^[17]).

References