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Blood, 1 May 2006, Vol. 107, No. 9, pp. 3417-3418.
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IMMUNOBIOLOGY
Comment on Evans et al, page 3593
Shedding of heterodimeric leukocyte integrin
Martin E. Hemler
DANA-FARBER CANCER INSTITUTE
Selective cleavage of the  L 2 integrin from neutrophils and monocytes during an in vivo human inflammatory response points to a novel mechanism for regulating leukocyte detachment.
Within the past 5 years, since the elucidation of the first integrin crystal structure,1 there has been extraordinary progress in understanding mechanisms by which integrin-mediated cell adhesion is turned on.2 However, there is considerably less understanding of integrin adhesion reversal and cell detachment. Regulated deactivation of integrins, "membrane ripping," and cytoskeletal rearrangements all may be involved. In some cases, an intracellular protease, calpain, helps to disassemble integrin-cytoskeletal complexes during cell detachment.3 On the extracellular side of the membrane, there have been occasional reports of single-chain integrin cleavage (involving either or chains alone). However, proteolytic cleavage and release of intact integrin heterodimers have not previously been observed under physiologic conditions. In this issue of Blood, Evans and colleagues now provide evidence for shedding of an active heterodimeric fragment of integrin L2 into the cell supernatant. This surprising result suggests a novel mechanism for cessation of integrin-dependent adhesion, to enable rapid detachment of leukocytes from substrate ICAM-1 on endothelium.
Cantharidin (Spanish fly) is a lipid-soluble blister beetle irritant that has been used for more than 100 years to promote skin blistering.4 This model of cutaneous inflammatory response in humans yields a substantial number of infiltrating leukocytes (0.1-10 x 106 cells/blister).
Evans et al noticed that expression of integrin L2 was markedly diminished on the surface of neutrophils and monocytes in blister fluid. Analysis of cell-free blister fluid revealed that membrane-bound integrin heterodimer (L, 180 kDa; 2, 90 kDa) had been converted to a soluble heterodimer of 110 kDa and 86 kDa, respectively (see figure). Meanwhile, a piece of the L subunit ( 70 kDa) was left behind on the cell. The observed shedding was remarkably specific on 3 levels. First, only L2 integrin, and not M2 or 41 integrins, was shed from neutrophils and monocytes. Second, L2 was not shed from lymphocytes present in the same blister fluid. Third, comparable L2 shedding was observed in nearly all (67 of 68) individuals treated with cantharidin but was not so obvious in another type of human inflammatory infiltrate. In synovial samples from arthritis patients, there was no evidence for decreased L2 on the surface of leukocytes, and only one of 15 samples showed biochemical evidence for conversion of L2 to its shed form.
As is typically the case for original research findings, many questions arise. For example, how is shedding achieved? Shedding activity was not conferred on cells by incubation with cell-free blister fluid, implying the existence of cis-acting proteases on the monocytes and neutrophils. Although only one protease is depicted in the figure, it is difficult to imagine a single protease cleaving both L, at a site approximately 10 to 15 nm above the membrane, and 2, at a site within 1 to 2 nm of the membrane. Also, why is this result obvious in the cantharidin blister model but not in other samples of inflammatory fluid? Evans et al suggest that in other clinical samples, shed L2 may be rapidly degraded and newly synthesized L2 may quickly replace that which was removed. However, another possibility is that cantharidin stimulation induces a unique profile of proteins, including proteases, on the surface of neutrophils and monocytes. In this regard, cDNA microarray analysis of cantharidin-treated HL-60 neutrophils revealed a wide range of specific gene expression changes.5 It will be important in future studies to assess the general relevance of L2 shedding. Available diagnostic anti-L monoclonal antibodies might be used in a 2-color flow cytometry screen of inflammatory infiltrates, potentially revealing the presence of leukocytes that have lost epitopes on the cleaved N-terminal part of L while retaining epitopes residing on the L stump, left behind after proteolysis. 
References
- Xiong JP, Stehle T, Diefenbach B, et al. Crystal structure of the extracellular segment of integrin alpha Vbeta3. Science. 2001;294: 339-345.[Abstract/Free Full Text]
- Ginsberg MH, Partridge A, Shattil SJ. Integrin regulation. Curr Opin Cell Biol. 2005;17: 509-516.[CrossRef][Medline]
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- Franco SJ, Huttenlocher A. Regulating cell migration: calpains make the cut. J Cell Sci. 2005;118: 3829-3838.[Abstract/Free Full Text]
- Benedek T. The cantharides blister and its application in microbiological research: a review of the literature and some suggestions. J Trop Med Hyg. 1939;42: 81-86.
- Zhang JP, Ying K, Xiao ZY, et al. Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarray. Int J Cancer. 2004;108: 212-218.[CrossRef][Medline]
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