Biology of immunoglobulin light chains

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Chapter

3

SECTION 1 - Immunoglobulin free light chains and their analysis

Biology of immunoglobulin light chains

Contents

Summary: Serum free light chains:-
  1. Contain constant region epitopes that are hidden in intact immunoglobulins.
  2. Are produced in excess by plasma cells.
  3. Have a half-life of a few hours because of rapid renal clearance.
  4. Bind to Tamm-Horsfall protein in the distal tubules and may obstruct urine flow.
  5. Are more frequently abnormal in serum than in urine because of renal metabolism.

3.1 Structure

Figure 3.1 An antibody molecule showing the heavy and light chain structure, together with free κ and λ FLCs.
Figure 3.2 Aκ FLC molecule showing the constant region (left), and the variable region (right) with its alpha helix (red). (Courtesy of J Hobbs)

Antibody molecules have a two-fold symmetry and are composed of two identical heavy and light chains, each containing variable and constant domains. The variable domains of each light chain/heavy chain pair combine to form an antigen-binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule. Light chains are of two types, κ and λ, and any given antibody molecule has either light chain but not both. There are approximately twice as many κ as λ molecules produced in humans but this is quite different in some mammals. Each FLC molecule contains approximately 220 amino acids in a single polypeptide chain that is folded to form the constant and variable region domains (Figures 3.1 and 3.2).

Domains are constructed from two β sheets. These are elements of protein structure made up of strands of the polypeptide chain (β strands) packed together in a particular shape. The sheets are linked by a disulfide bridge and together form a roughly barrelshaped structure known as a β barrel (Figure 3.2).

The constant (C) domains of the FLCs show little variation except for the amino acid substitutions found in the Km allotypes on κ molecules and the Oz and Kern isotypes on λ molecules. In contrast, the variable (V) domain has huge structural diversity, particularly in association with the antigen binding amino acids. In addition, the first 23 amino acids of the 1st variable domain framework region have a limited number of variations known as subgroups. Using monoclonal antibodies, 4 kappa (Vκ1 -Vκ4) and 6 lambda subgroups (Vλ 1 -Vλ 6) can be identified [1]. The specific subgroup structures influence the potential of the FLCs to polymerise such that AL amyloidosis is associated with Vλ6 and LCDD with Vκ1 and Vκ4.

3.2 Synthesis

Figure 3.3 Construction of light chains.
Figure 3.4 Development of the B-cell lineage and associated diseases.

κ FLC molecules (chromosome 2) are constructed from approximately 40 functional Vκ gene segments, five Jκ gene segments and a single Cκ gene. λ molecules (chromosome 22) are constructed from about 30 Vκ gene segments and four pairs of functional Jλ gene segments and a Cλ gene (Figure 3.3).

FLCs are incorporated into immunoglobulin molecules during B lymphocyte development and are expressed initially on the surface of pre B-cells. Production of FLCs occurs throughout the rest of B-cell development and in plasma cells, where secretion is highest. Tumours associated with the different stages of B-cell maturation will secrete monoclonal FLCs into the serum where they may be detected by FLC immunoassays (Figure 3.4 and Chapter 18 ).

3.3 Production

Production of FLCs in normal individuals is approximately 500mg/day from bone marrow and lymph node cells [1][2]. The molecules enter the blood and are rapidly partitioned between the intra-vascular and extra-vascular compartments. The normal plasma cell content of the bone marrow is about 1% while in MM the plasma cell content can rise to over 90%. The bone marrow may contain 5-10% plasma cells in chronic infections and autoimmmune diseases and this is associated with hypergammaglobulinaemia and corresponding increases in polyclonal sFLC concentrations. Bone marrow identification of monoclonal plasma cells by histology is an essential part of MM diagnosis and is frequently based on identifying intracellular κ and λ by direct immunofluorescence techniques (Figure 3.5A).

Plasma cells produce one of five heavy chain types together with κ or λ molecules. There is approximately 40% excess FLC production over heavy chain synthesis to allow proper conformation of the intact immunoglobulin molecules. As already indicated, there are twice as many κ producing plasma cells as λ plasma cells. κ FLCs are normally monomeric, while λ FLCs tend to be dimeric, joined by disulphide bonds but higher polymeric forms of both FLCs may occur (Figure 3.5B).

3.4 Clearance and metabolism

Figure 3.5 A. Immunohistochemical staining of κ producing bone marrow plasma cells from a patient with MM using fluorescein-conjugated, anti-κ antiserum. 5 plasma cells can be seen. B. Diagrammatic representation of plasma cells producing intact immunoglobulins with monomeric κ and dimeric λ FLC molecules.
Figure 3.6 Nephron showing filtration, metabolism and excretion of FLCs. (Courtesy of R Johnson and J Feehally).
Figure 3.7 A. Waxy cast from the urine of a patient with multiple myeloma. (Courtesy of R Johnson and J Feehally).B. Monoclonal FLC casts in the distal tubules of a patient with myeloma kidney. (Courtesy of C Hutchison).
Figure 3.8 Changes in serum and urine free light chain concentrations during the evolution of a hypothetical patient with light chain multiple myeloma.

In normal individuals, sFLCs are rapidly cleared and metabolised by the kidneys depending upon their molecular size. Monomeric FLCs, characteristically κ, are cleared in 2-4 hours at 40% of the glomerular filtration rate. Dimeric FLCs, typically λ, are cleared in 3-6 hours at 20% of the glomerular filtration rate, while larger polymers are cleared more slowly. Removal may be prolonged to 2-3 days in MM patients in complete renal failure (Chapter 13) [1][2][3]. In contrast, IgG has a half-life of 21 days.

Figure 3.6 shows a nephron, of which there are approximately half a million in each kidney. Each nephron contains a glomerulus with basement membrane fenestrations that allows filtration of serum molecules into the proximal tubules. The pore sizes are variable with a restriction in filtration commencing at about 20-40kDa and being complete by 60kDa. Protein molecules that pass through the glomerular pores are then either absorbed unchanged (such as albumin) or degraded in the proximal tubular cells and absorbed or excreted as fragments [4]. This megalin/cubulin absorption pathway is important and is designed to prevent loss of large amounts of proteins and peptides into urine. It is very efficient and can process between 10-30g of small molecular weight proteins per day, so under normal conditions, none passes beyond the proximal tubules [5][6][7].

The distal tubule secretes large amounts of uromucoid (Tamm-Horsfall protein). This is the dominant protein in normal urine and is thought to be important in preventing ascending urinary infections. It is a relatively small glycoprotein (80kDa) that aggregates into polymers of 20-30 molecules. Interestingly, it contains a short peptide motif that specifically binds FLCs [8]. Together they form waxy casts that are characteristically found in acute renal failure associated with LCMM (Figures 3.7A, 3.7B and Chapter 13) [9][10].

In normal individuals, 1-10mg of FLCs is excreted per day into the urine. Its exact origin is unclear but it probably enters the urine via the mucosal surfaces of the distal part of the nephrons and the urethra, alongside secretory IgA. This secretion is part of the mucosal defence system that prevents infectious agents entering the body. The 500mg of FLCs produced per day by the normal lymphoid system, therefore, flow through the glomeruli and are completely processed by the proximal tubules [4].

If the proximal tubules of the nephrons are damaged or stressed (such as in hard exercise), filtered FLCs may not be completely metabolised. Small amounts may then enter the urine. Although important as markers of general renal function and glomerular function in particular, serum creatinine and cystatin C measurements cannot detect such minor degrees of nephron dysfunction.


As noted above, κ FLC monomers are cleared three times faster than dimeric λ molecules because of their smaller size [11]. Although κ production rates are twice that of λ, its faster removal ensures that the actual serum concentrations are approximately 50% lower (Chapter 5 and 20).

Because of the huge proximal tubule metabolism, the amounts of FLCs in urine, even when production is considerably increased, are more dependent upon renal function than synthesis by the tumour. As a consequence, serum and urine FLC concentrations may not be similar during the evolution of LCMM. This is shown in a hypothetical patient in Figure 3.8. The red line shows the steady increase in sFLCs as the tumour grows over the first 12 months. When synthesis of FLCs exceeds 10-30g/day (greater than 30 times normal) there is an overflow proteinuria and large amounts of FLCs enter the urine. This is normally when patients with LCMM are identified.

The FLCs that overwhelm the proximal tubules’ absorption mechanisms enter the distal tubules and may cause inflammation or precipitate as casts. These can block the flow of urine causing the death of the respective nephrons (Figures 3.7A and 3.7B and Chapter 13 and 20). Rising concentrations of sFLCs are filtered by the remaining nephrons leading to a vicious cycle of accelerating renal damage with further increases in sFLCs.

This process lengthens the serum half-life of the FLCs so that concentrations rise rapidly, in contrast to urine excretion, which falls as the patients develop terminal renal failure and is obviously zero when patients become aneuric (Figure 3.8). Consequently, the levels of serum and urine FLCs diverge during the later stages of the disease. While increasing serum concentrations indicate disease progression, falling urine concentrations may falsely suggest disease stabilisation or improvement. Thus, Nowrousian et al., showed that urine FLC excretion decreased at high sFLC concentrations if there was significant renal impairment. In a similar manner, urine concentrations may increase as renal function improves with treatment of the tumour.

Understanding the toxic effects of FLCs on renal function and FLC concentrations in serum and urine is important. The inevitable conclusion, from the physiological and pathological mechanisms described above, is that serum is preferable to urine for assessing FLC concentrations in patients with monoclonal FLC diseases.

Test Questions
  1. Is Bence Jones proteinuria “overflow”, “glomerular” or “tubular” in origin?”
  2. What are the normal serum half-lives of IgG and FLCs?
  3. Serum albumin concentrations are reduced in patients with nephrotic syndrome with gross proteinuria. Are serum FLC concentrations also reduced in these circumstances?
  4. Which protein binds FLCs in the distal tubules?
  5. Do urine FLC concentrations always increase alongside rising serum FLC concentrations?


Chapter 2 Back to Contents Page Chapter 4

References

  1. ↑ 1.0 1.1 1.2 Solomon A. Light chains of human immunoglobulins. Methods Enzymol 1985; 116: 101 – 21 PMID: 3937021
  2. ↑ 2.0 2.1 Waldmann TA, Strober W, Mogielnicki RP. The renal handling of low molecular weight proteins. II. Disorders of serum protein catabolism in patients with tubular proteinuria, the nephrotic syndrome, or uremia. J Clin Invest 1972; 51: 2162 – 74 PMID: 5054468
  3. ↑ Miettinen TA, Kekki M. Effect of impaired hepatic and renal function on Bence Jones protein catabolism in human subjects. Clin Chim Acta 1967; 18: 395 - 407
  4. ↑ 4.0 4.1 Russo LM, Bakris GL, Comper WD. Renal handling of albumin: a critical review of basic concepts and perspective. Am J Kidney Dis 2002; 39: 899 – 919 PMID: 11979334
  5. ↑ Abraham GN, Waterhouse C. Evidence for defective immunoglobulin metabolism in severe renal insufficiency. Am J Med Sci 1974; 268: 227 – 33 PMID: 4217565
  6. ↑ Wochner RD, Strober W, Waldmann TA. The role of the kidney in the catabolism of Bence Jones proteins and immunoglobulin fragments. J Exp Med 1967; 126: 207 – 21 PMID: 4165739
  7. ↑ Maack T, Johnson V, Kau ST, Figueiredo J, Sigulem D. Renal filtration, transport, and metabolism of low-molecular-weight proteins: a review. Kidney Int 1979; 16: 251 – 70 PMID: 393891
  8. ↑ Ying WZ, Sanders PW. Mapping the Binding Domain of Immunoglobulin Light Chains for Tamm-Horsfall protein. Am J Path 2001; 158: 1859-1866 PMID: 11337384.
  9. ↑ Sanders PW, Booker BB, Bishop JB, Cheung HC. Mechanisms of intranephronal proteinaceous cast formation by low molecular weight proteins. J Clin Invest 1990; 85: 570 - 6 PMID2298921
  10. ↑ Sanders PW, Booker BB. Pathobiology of cast nephropathy from human Bence Jones proteins. J Clin Invest 1992; 89: 630 – 9 PMID: 1737851
  11. ↑ Arfors KE, Rutili G, Svensjo E. Microvascular transport of macromolecules in normal and inflammatory conditions. Acta Physiol Scand Suppl 1979; 463: 93 – 103 PMID: 382749
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