Biology of immunoglobulin light chains

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Chapter

3

SECTION 1 - Introduction

Biology of immunoglobulin light chains

Contents

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

3.1. Structure

antibody structure showing free light chain epitopes
Figure 3.1. An antibody molecule showing the heavy and light chain structure, together with free κ and λ FLCs.
Ribbon diagram of a kappa free light chain molecule
Figure 3.2. A κ 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 in any given antibody molecule only one type occurs. Approximately twice as many κ as λ molecules are produced in humans but in other mammals this ratio can vary. Each free light chain (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 barrel-shaped structure known as a β barrel (Figure 3.2).

Genes that encode antibodies are assembled in B lymphocytes by joining multiple gene segments that are far apart in germline DNA. The light chain variable domain is constructed from V and J gene segments, whilst the constant domain is encoded by a separate C gene segment (Figure 3.3). There are multiple copies of the gene segments in germline DNA, and a random selection of individual V, J and C genes contributes to the diversity of immunoglobulin light chains.

The constant domains of light chains show little amino acid sequence variation. The human genome contains a single κ constant gene for which three serologically-defined allotypes have been defined, designated Km1, Km2 and Km3 [1]. These allotypes define three km alleles that differ in two amino acids, as presented in table Table 3.1. The genome also contains a variable number of λ constant genes, giving rise to multiple lambda chain isotypes. These λ isotypes can be distinguished serologically through the expression of Mcg, Kern and Oz markers. For example, the protein product of the IGLC1 gene is Mcg+Kern+Oz-. There is no evidence that these variants affect FLC measurements using Freelite® assays, which are based on polyclonal antisera (see Chapter 4).

Km allele
Amino Acids
KM1 Val(153) Leu(191)
KM1,2 Ala(153) Leu(191)
KM3 Ala(153) Val(191)

Table 3.1. Amino acid substitutions in κ constant domains.

In contrast, the variable domains of light chains exhibit 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 (Vλ1 - Vλ6) can be identified [2]. The specific subgroup structures influence the potential of the FLCs to polymerise such that AL amyloidosis is associated with Vλ6 and light chain deposition disease (LCDD) with Vκ1 and Vκ4.

3.2. Synthesis

DNA recombination of V, J and C light chain gene segments
Figure 3.3. Construction of light chains.
B cell development from lymphoid progenitors to plasma cells
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 FLC molecules (chromosome 22) are constructed from approximately 30 Vλ gene segments, and four (or more) pairs of functional Jλ gene segments and Cλ genes.

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

In normal individuals, approximately 500mg of FLCs are produced each day from bone marrow and lymph node cells [2][3]. The molecules enter the blood and are rapidly partitioned between the intravascular and extravascular compartments. The normal plasma cell content of the bone marrow is about 1%, whereas in multiple myeloma (MM) this 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 or flow cytometry 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 mentioned previously, there are twice as many κ-producing plasma cells as λ-producing plasma cells. κ FLCs are normally monomeric, while λ FLCs tend to be dimeric, joined by disulphide bonds, however higher polymeric forms of both FLCs may occur (Figure 3.5B).

3.4. Clearance and metabolism

Bone marrow immunohistochemistry and plasma cell diagram
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.
Nephron diagram
Figure 3.6. Nephron showing filtration, metabolism and excretion of FLCs. (This figure was published in Acute renal failure: Myeloma kidney. Winearls CG. In: Johnson RJ, Feehally J, eds. Comprehensive clinical nephrology, Mosby: Page 238, figure 17.5 © Elsevier (2003)).
Urine containing cast and renal biopsy showing myeloma kidney
Figure 3.7. A. Waxy cast from the urine of a patient with multiple myeloma. (This figure was published in Investigation of renal disease: Urinalysis. Fogazzi GB. In: Johnson RJ, Feehally J, eds. Comprehensive clinical nephrology, Mosby: Page 41, figure 4.3B © Elsevier (2003)). B. Monoclonal FLC casts in the distal tubules of a patient with myeloma kidney. (Courtesy of C Hutchison).
evolution of light chain multiple myeloma
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) [2][3][4][5]. In contrast, IgG has a half-life of 21 days with minimal renal clearance (Chapter 10).

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, which allow 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 (as with albumin) or degraded in the proximal tubular cells and absorbed or excreted as fragments [6]. 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 [7][5][8].

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 [9]. Together they form waxy casts that are characteristically found in acute renal failure associated with light chain multiple myeloma (LCMM) (Figures 3.7A, 3.7B and Chapter 13) [10][11].

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 each day by the normal lymphoid system therefore flow through the glomeruli and is completely processed by the proximal tubules [6].

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

As noted above, due to their smaller size κ FLC monomers are cleared 2-3 times faster than dimeric λ molecules [12]. Although κ production rates are twice that of λ, their faster removal ensures that the actual serum concentrations are approximately 50% lower (Chapter 5 and 20).

Because of the huge metabolic capacity of the proximal tubule, the amount of FLCs in urine, even when production is considerably increased, is 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. It is normally at this point that patients with LCMM are identified.

When FLCs overwhelm the proximal tubules’ absorption mechanisms, they enter the distal tubules and may cause inflammation or precipitate as casts. This can block the flow of urine causing affected nephrons to perish (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 sFLC concentrations.

This process lengthens the serum half-life of FLCs so that concentrations rise rapidly as urine excretion decreases with the onset of terminal renal failure, falling to zero as the patient becomes aneuric (Figure 3.8). Consequently, the amounts of FLCs in serum and urine diverge during disease progression. While increasing serum concentrations indicate worsening disease, falling urine concentrations may falsely suggest disease stabilisation or improvement. For example, Nowrousian et al. showed that urine FLC excretion decreased at high sFLC concentrations when there was significant renal impairment [13]. In contrast, successful treatment of tumours may lead to a reduction of tubular casts, increased urine flow and more FLCs in urine.

Understanding the nephrotoxicity of FLCs and how it can influence 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.

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 sFLC concentrations also reduced in these circumstances?
  4. Which protein binds FLCs in the distal tubules?
  5. Do urine FLC concentrations always increase alongside rising sFLC concentrations?


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References

  1. Jefferis R, Lefranc MP. Human immunoglobulin allotypes: possible implications for immunogenicity. MAbs 2009;1:332-8 PMID:20073133
  2. 2.0 2.1 2.2 Solomon A. Light chains of human immunoglobulins. Methods Enzymol 1985;116:101-21 PMID:3937021
  3. 3.0 3.1 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
  4. 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 PMID:3937021
  5. 5.0 5.1 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
  6. 6.0 6.1 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
  7. Abraham GN, Waterhouse C. Evidence for defective immunoglobulin metabolism in severe renal insufficiency. Am J Med Sci 1974;268:227-33 PMID:4217565
  8. 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
  9. 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
  10. 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 PMID:2298921
  11. Sanders PW, Booker BB. Pathobiology of cast nephropathy from human Bence Jones proteins. J Clin Invest 1992;89:630-9 PMID:1737851
  12. 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
  13. Nowrousian MR, Brandhorst D, Sammet C, Kellert M, Daniels R, Schuett P, et al. Serum free light chain analysis and urine immunofixation electrophoresis in patients with multiple myeloma. Clin Cancer Res 2005;11:8706-14 PMID:16361557

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