Shi Shuiliang, Ciurli Cristina, Cartman Annie, Pidoux Isabelle, Poole A Robin, Zhang Yiping
Shriners Hospitals for Children, Montreal, Quebec, Canada.
Arthritis Rheum. 2003 Oct;48(10):2903-15. doi: 10.1002/art.11270.
Experimental immunity to the G1 domain of the cartilage proteoglycan (PG) aggrecan (AG1) leads to the development of spondylitis as well as polyarthritis in BALB/c mice. The PG versican contains a structurally similar G1 domain (VG1). This study was conducted to determine whether immunity to VG1 would elicit similar pathology in these mice.
Recombinant natively folded VG1 and AG1 were prepared. BALB/c mice received either a series of 5 injections of human VG1 or AG1, or no protein. Polyarthritis was determined clinically, and spondylitis and sacroiliitis histologically. Immunohistochemistry of rat tissues was used to study the localization of versican. Enzyme-linked immunosorbent assays were employed to study humoral immunity to the recombinant proteins as well as to overlapping synthetic peptides covering all these human G1 domains and mouse homologs. Affinity-purified antibodies to human AG1 and VG1 were isolated from sera of hyperimmunized mice. T lymphocyte proliferation assays were performed using recombinant human proteins. T cell lines reactive with specific immunodominant T cell epitopes in human AG1 and VG1 were isolated. Synthetic peptides encoding sequences in these human proteins and in corresponding mouse proteins were used in these analyses. Guanidinium chloride extracts of mouse spines were also used in Western blots to study antibody cross-reactivity.
Immunity to recombinant VG1 did not result in clinical polyarthritis. There was, however, clear evidence that VG1, like AG1, could induce spondylitis in the lumbar spine and sacroiliitis. Accumulation of mononuclear cells was observed in spinal ligaments adjacent to the intervertebral disc, in the intervertebral disc, and in the sacroiliac joints, the same sites where versican is localized. In contrast to AG1-immunized mice, in which T cells reactive with human AG1 cross-reacted with mouse AG1, there was no evidence in VG1-immunized mice that T cell immunity to human VG1 was cross-reactive with a mouse synthetic peptide that contained the sequence corresponding to the single immunodominant T cell sequence recognized in human VG1. Antibodies to specific sequences in human VG1 did, however, cross-react with human AG1 and with corresponding peptide sequences in mouse versican and aggrecan and with mouse proteins containing VG1 and AG1, present in mouse spine extracts. Similarly, antibodies to human AG1 cross-reacted with human VG1 and with extracted mouse VG1 and AG1 and synthetic peptides containing mouse sequences that corresponded to the reactive human epitopes in AG1 and VG1.
These observations suggest that humoral immunity to human VG1 is involved in the induction of experimental spondylitis and sacroiliitis in BALB/c mice. This humoral immunity is cross-reactive with mouse versican and aggrecan but is not associated with polyarthritis, probably because of the lack of cross-reactive T cell immunity and the absence of detectable versican in articular cartilage limbs. Induction of polyarthritis by bovine or human aggrecan requires the involvement of immunity mediated by T lymphocytes that are cross-reactive to a mouse aggrecan epitope. Together these observations suggest that humoral immunity to versican as well as immunity to aggrecan may be of importance in the development of the spinal pathology characteristic of spondylarthropathies.
对软骨蛋白聚糖（PG）聚集蛋白聚糖（AG1）的G1结构域进行实验性免疫会导致BALB/c小鼠发生脊柱炎和多关节炎。PG多功能蛋白聚糖含有结构相似的G1结构域（VG1）。本研究旨在确定对VG1的免疫是否会在这些小鼠中引发类似的病理变化。
制备重组天然折叠的VG1和AG1。BALB/c小鼠接受5次人VG1或AG1注射，或不注射蛋白质。临床确定多关节炎，组织学确定脊柱炎和骶髂关节炎。使用大鼠组织的免疫组织化学研究多功能蛋白聚糖的定位。采用酶联免疫吸附测定法研究对重组蛋白以及覆盖所有这些人G1结构域和小鼠同源物的重叠合成肽的体液免疫。从超免疫小鼠的血清中分离出亲和纯化的抗人AG1和VG1抗体。使用重组人蛋白进行T淋巴细胞增殖测定。分离出与人AG1和VG1中特定免疫显性T细胞表位反应的T细胞系。编码这些人蛋白和相应小鼠蛋白序列的合成肽用于这些分析。小鼠脊柱的氯化胍提取物也用于蛋白质印迹，以研究抗体交叉反应性。
对重组VG1的免疫未导致临床多关节炎。然而，有明确证据表明，与AG1一样，VG1可诱导腰椎脊柱炎和骶髂关节炎。在椎间盘相邻的脊柱韧带、椎间盘和骶髂关节中观察到单核细胞聚集，多功能蛋白聚糖也定位在这些部位。与用AG1免疫的小鼠不同，在用AG1免疫的小鼠中，与人AG1反应的T细胞与小鼠AG1发生交叉反应，而在用VG1免疫的小鼠中，没有证据表明对人VG1的T细胞免疫与包含与人VG1中识别的单一免疫显性T细胞序列相对应序列的小鼠合成肽发生交叉反应。然而，针对人VG1中特定序列的抗体确实与人AG1以及小鼠多功能蛋白聚糖和聚集蛋白聚糖中的相应肽序列以及小鼠脊柱提取物中存在的含有VG1和AG1的小鼠蛋白发生交叉反应。同样，抗人AG1抗体与人VG1以及提取的小鼠VG1和AG1以及含有与AG1和VG1中反应性人表位相对应的小鼠序列的合成肽发生交叉反应。
这些观察结果表明，对人VG1的体液免疫参与了BALB/c小鼠实验性脊柱炎和骶髂关节炎的诱导。这种体液免疫与小鼠多功能蛋白聚糖和聚集蛋白聚糖发生交叉反应，但与多关节炎无关，可能是因为缺乏交叉反应性T细胞免疫且关节软骨肢体中未检测到多功能蛋白聚糖。牛或人聚集蛋白聚糖诱导多关节炎需要由对小鼠聚集蛋白聚糖表位发生交叉反应的T淋巴细胞介导的免疫参与。这些观察结果共同表明，对多功能蛋白聚糖的体液免疫以及对聚集蛋白聚糖的免疫可能在脊柱关节病特征性的脊柱病理发展中起重要作用。
