Journal of Biological Regulators and Homeostatic Agents

Immunophenotypic peculiarities of mobilized stem (CD34+) cells in blood from patients with severe spinal cord injury

N.N TUPITSYN, V.N. YARYGHIN, AS. BRYUKHOVETSKIY, L.YU. GRIVTSOVA , G.L MENTKEVICH, I.S. DOLGOPOLOW, A.YU. ZAITSEV. M.I. DAVYDOV

ABSTRACT.Immunophenotype of mobilized stem blood cells (CD34*) was studied in 29 patients with late post-traumatic spinal lesions. The CD34* cells demonstrated different levels of expression of CD45, CD38, monomorphic determinants HLA-DR and gp130 epitopes. Most patients presented with a CD34* cell fraction with no or low expression of common leukocytic antigen CD45. Only 2 patients had >15% of HLA-DR-CD38- cells in the CD34* fraction. A common transducer molecule of interleukin-6 family cytokines gp130 was expressed on stem (CD34*) cells in all the cases, 26% of the patients had an activated gp130 phenotype, i.e. a combination of C7* and A1- epitopes.

KEY WORDS: Spinal cord injury, CD34, CD45, gp130 epitopes

Introduction

Mobilized autologous stem cell (MASC) intrathe­cal administration is a new treatment approach in patients with a spinal cord injury. We have recently demonstrated (1) that response as recovery or improvement in paretic limb mobility, improvement m pelvic organ function, and partial, mosaic improvement in sensibility was detected in 61% of the cases. Clinical findings were confirmed by a complex urodynamical study and electromyography MASC grafting, and mechanism of innervation recovery (improvement) due to intrathecal adminis­tration of stem cells is unknown. Autologous mobi­lized hemopoietic stem cell grafting is commonly used in oncology and oncohematology. The method consists of administration of a blood mononuclear fraction enriched with CD34* cells as obtained by leuKapheresis to restore chemotherapy-depressed hemopoiesis. Such a "substitutive" mechanism of action, i.e. generation of neuronal or glial cells from mobilized CD34* stem cells after their intrathecal administration to patients with spinal injury, seems unlikely though cannot be definitely excluded. Mobilization of stem (CD34-) cells into peripheral circulation under the effect of colony stimulating fac­tors is a principal requirement for the treatment approach in question (these mobilization and CD34 appearance/elevation were confirmed in all cases studied). For this reason we focused first of all on detailed study of CD34-positive stem cells, i.e. MASC, in this paper, while not ruling out in principle the role of CD34-negative fraction within the intrathe­cal^ grafted cell population. Detailed characterization of qualitative composi­tion of the CD34* MASC from the standpoint of their possible nonhemopoietic commitment may be a key to this problem. To study this possibility we analyzed expression of CD45 molecules and coexpression of HLA-DR and CD38 on CD34* MASC. We also stud­ied expression and activation of receptor gp130, a transducer molecule of interleukin-6 (IL-6) cytokine family, on CD34* cells. The gp130 is a hemopoietic and non-hemopoietic stem cell coreceptor needed for realization of proliferative and differentiating activities of the following cytokines: IL-6, ciliary neu­rotrophic factor (CNTF), leukemia-inhibiting factor (LIF). oncostatin M (OM), cardiotrophin-1 (CT-1) and IL-11 (2-4). Activation of gp130 under the effect of any of these cytokines may be assessed by gp130 epitope structure (5).

Materials and Methods

The study was performed in 29 patients in late spinal cord post-traumatic period (in most cases - 3 to 10 years after injury). The patient age was rang- ing from 17 to 65 years (median age 30 years, only patients were above 50); there were 24 males and females. Autologous stem cell mobilization and harvesting was performed by standard techniques recommend­ed and approved by the European Bone Marrow Transplantation Society (6). Recombinant human G-CSF (filgastrim - Neupogen) given for 4 days twice a day at dosage of 2.5 u./kg-6.8 u/kg (mean - 4.3 uVkg) was used as a colony stimulating factor. Stem cells and their subpopulations were deter­mined in leukapheresis product according to guide­lines provided Keeney et al. (7) by direct immunoflu-orescent staining of cells with subsequent flow cytometry. To measure CD34* cell number we used in all cases PE-labelled monoclonal antibodies (mAbs) HPCA-2 (BD, USA) and mouse lgG1-PE as a control. In study of CD34 subpopulation PE/Cy5 or FITC-conjugated HPCA-2 were used as well. Expression of CD45 on mobilized CD34* cells was studied in all 29 patients, we used FITC-labelled anti-CD45 conjugate (DakoCytomation, Denmark). To study monomorphic HLA-DR and CD38 on mobi­lized stem (CD34*) cells corresponding PE- or FITC-mAb conjugates were used (DakoCytomation, Denmark). Monoclonal antibodies to transducer molecule gp130 were kindly given by Jean Brochier (INSERM, France). Two epitopes of gp130 were studied: A1 - at dimerization site and C7 at another functionally active domain. Conjugates of these antibodies with PE/Cy5 (for A1) and PE (for C7) were acquired together with R. Jones (HMDS, Leeds, UK). Acquisition and analysis of data was made using a FACScan (Becton Dikinson, USA) flow cytometer. Stem cell subpopulations were studied in the CD34* cell gate with low laser ray scattering characteristics (low SSC) similar to those for lymphoid cells. Subsequent analysis was performed on PC with the program WinMDI.

Results

Absolute number of CD34+ cells collected (just 1 apheresis procedure) in patients was in the range from 1.4 to 59.1 x107, mean - 12.7 x 107. The num­ber of infused CD34* MASC in average was about 5.3 x 106 per each intratecal transfusion (1). In 21 of 29 patients with spinal injury within mobi­lized peripheral blood stem (CD34+) cells there was noted a distinctive cell population with a very low (below the level of expression on granulocytes) or no CD45 expression. Percentage of CD45- cells among CD34* ones was low (5-18.9%) in 5 (17%), moder­ate (24-49%) in 10 (34%) and high (62-98%) in 6 (21%) patients. Eight patients had no CD45-negative fraction in their CD34+ population. Fig. 1 shows low or no expression of CD45 on 94% of CD34+ cells

with a CD45- fraction easily seen. There were some­what inverse correlation (statistically not significant, R=-0.307, p=0.105) between the percentage of CD34* cells in apheresis product and the percent­age of CD45- cells within CD34* cells.Detailed study of CD38 and HLA-DR expression on mobilized CD34* blood cells was performed in 12 patients with spinal injury. Proportion of CD38HLA-DR- cells in the CD34* population was small (less than 9% in 10 patients) and reached 16 and 23% in 2 cases only (figure 2). In both cases with a large HLADR-CD38- proportion in CD34+ stem cells there was no CD45 expression on most CD34* cells as shown in the case in Fig. 2.

Two epitopes of gp130 molecule, i.e. a functional epitope C7 participating in LIF and OM signal trans­duction and a functional epitope A1 involved in gp130 dimerization under the effect of any of the IL-6 family cytokines (2) were analyzed on mobilized stem cells in 15 patients with spinal injuries. There were different levels of gp130 expression on CD34* cells: epitope C7 was present on all CD34* cells in 13 cases, 9 of them had epitope A1 coexpression which was evidence of gp130 being not activated (5). In four patients C7 epitope monomorphous expression on stem cells was noted, while epitope A1 expression was fully absent or found on some cells only. This epitopic structure is indicative of gp130 molecule activation under the effect of certain IL-6-family cytokines (5). Two cases presented with epitope A1 expression and practically full absence of epitope C7. Such a combination of membrane gp130 epitopes has not been described in hemato­logical systems (normal and malignant blood cells, hemopoietic stem cells) so far, and theoretically may be indicative of initial stages of LIF and OM signaling yet failing to lead to gp130 dimerization. Types of gp130 expression are given in Fig. 3.

Discussion

In the present study we were attempted at study of some immunophenotypic peculiarities of MASC in patients with spinal cord injury. Standard mobiliza­tion procedure was quite effective to collect enough CD34* cells for intrathecal infusions. Antigen CD45 is expressed on all blood cells such as immature precursors, lymphocytes, granulo­cytes etc. except erythrocytes. The CD45 expres­sion is usually increasing as the leucocytes are mat­urating. Haematopoietic stem cells (CD34+) may have different levels of CD45 expression ranging from slight to marked expression. The presence of CD45 on stem (CD34*) hematopoietic blood and bone marrow cells is the basis of a best known pro­tocol for determination of these cells, i.e. ISHAGE (8). Our findings demonstrate that mobilized stem (CD34*) cells from blood of patients with spinal injury have different patterns of common leukocyte antigen CD45 expression. Most patients presented with a distinct CD45-negative cell fraction (CD34+CD45-) in the MASC population - the finding which is very rarely seen in haematopoietic stem cells. CD34 expression is characteristic of both haematopoietic and stromal precursors that are a small percentage within CD34+CD38-HLA-DR- frac­tion (9). According to the published data it is these cells that may potentially differentiate in a non-hematopoietic lineage (9). Our data did not confirm the existence of prominent proportion of CD38-HLA-DR- cells within MASC in patients with spinal injury. There are no techniques to directly verify neu­ronal or glial commitment of mobilized stem cells so far. It is possible that CD45negative MASC may acquire non-hemopoietic differentiation in a specific microenvironment following intrathecal administra­tion, while according to modern concepts such course of events is absolutely impossible for CD45*CD34+ cells. We believe that study of possibility for stem cells to differentiate in neuronal or glial lineage under the direct cytokine-free action on gp130 is the most promising approach to verify the hypothesis of the MASC substitutive role. We demonstrated a similar mechanism for hematopoietic stem cells previously (3,10), though it was not studied in neuronal precur­sors. That is why we also focused in our study on measurement of expression and activation of gp130, a transducer receptor of IL-6 family cytokines. To our knowledge there were no such studies so far. Different patterns of gp130 expression and activa­tion were noted, some of them being not typical for haematopoietic stem cells. Our findings confirm gp130 to be expressed on MASC (CD34*) both in non-activated (epitopes A1+C7*) and activated (A1-C7+) states. Besides, there were 2 cases with a practically complete absence of epitope C7 which theoretically might occur at initial OM and LIF signal­ing stages. 4 We did not attempt to assess MASC treatment efficacy in patients with spinal cord injury with respect to the presence of CD45negative cells or gp130 activation status. Clinical response was detected also in patients having no CD45-negative MASC. This may suggest that therapeutic response was induced not only by CD34+CD45- MASC but also by CD34-negative or CD34+CD45* cells as administered simultaneously. The MASC substitute mechanism of action seems to be most prone to crit­icism and difficult to prove. It is evident that induction of nerve process growth, myelinization, angiogenesis and many other factors also play an important role in regeneration and improvement of tissue innervation under the effect of MASC.

References

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