Potential role for hyaluronan and the hyaluronan receptor RHAMM in mobilization and trafficking of hematopoietic progenitor cells.

Although the mechanism(s) underlying mobilization of hematopoietic progenitor cells (HPCs) is unknown, detachment from the bone marrow (BM) microenvironment and motility are likely to play a role. This work analyzes the motile behavior of HPCs and the receptors involved. CD34(+)45(lo/med)Scatterlo/med HPCs from granulocyte colony-stimulating factor (G-CSF)-mobilized blood and mobilized BM were compared with steady-state BM for their ability to bind hyaluronan (HA), their expression of the HA receptors RHAMM and CD44, and their motogenic behavior. Although RHAMM and CD44 are expressed by mobilized blood HPCs, function blocking monoclonal antibodies (MoAbs) identified RHAMM as a major HA binding receptor, with a less consistent participation by CD44. Permeabilization of mobilized blood HPCs showed a pool of intracellular (ic) RHAMM and a smaller pool of icCD44. In contrast, steady-state BM HPCs have significantly larger pools of icRHAMM and icCD44. Also, in contrast to mobilized blood HPCs, for steady-state BM HPCs, MoAbs to RHAMM and CD44 act as agonists to upregulate HA binding. The comparison between mobilized and steady-state BM HPCs suggests that G-CSF mobilization is associated with depletion of intracellular stores of HA receptors and modulates HA receptor usage. To confirm that mobilization alters the HA receptor distribution and usage by HPCs, samples of BM were collected at the peak of G-CSF mobilization in parallel with mobilized blood samples. HA receptor distribution of mobilized BM HPCs was closely matched with mobilized blood HPCs and different from steady-state BM HPCs. Mobilized BM HPCs had lower pools of icHA receptors, similar to those of mobilized blood HPCs. Treatment of mobilized BM HPCs with anti-RHAMM MoAb decreased HA binding, in contrast to steady-state BM HPCs. Thus, G-CSF mobilization may stimulate an autocrine stimulatory loop for HPCs in which HA interacts with basal levels of RHAMM and/or CD44 to stimulate receptor recycling. Consistent with this, treatment of HPCs with azide, nystatin, or cytochalasin B increased HA binding, implicating an energy-dependent process involving lipid rafts and the cytoskeleton. Of the sorted HPCs, 66% were adherent and 27% were motile on fibronectin plus HA. HPC adherence was inhibited by MoAbs to beta1 integrin and CD44, but not to RHAMM, whereas HPC motility was inhibited by MoAb to RHAMM and beta1 integrin, but not to CD44. This finding suggests that RHAMM and CD44 play reciprocal roles in adhesion and motility by HPCs. The G-CSF-associated alterations in RHAMM distribution and the RHAMM-dependent motility of HPCs suggest a potential role for HA and RHAMM in trafficking of HPCs and the possible use of HA as a mobilizing agent in vivo.

A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.

In multiple myeloma (MM), the VDJ rearrangement of the immunoglobulin heavy chain expressed by MM plasma cells provides a unique clonotypic marker. Although clonotypic MM cells have been found in the circulation, their number has been controversial. Our objective was to provide direct evidence, using single-cell assays, for the frequency of clonotypic cells in blood of 18 MM patients, and to confirm their identity as B cells. The clonotypic Ig heavy-chain (IgH) VDJ was determined from single plasma cells using consensus reverse transcriptase-polymerase chain reaction (RT-PCR), subcloning, and sequencing. For all patients, using patient-specific primers, clonotypic transcripts were amplified from 10 or more individual plasma cells. Using in situ RT-PCR, for all patients greater than 80% of plasma cells were found to be clonotypic. Three separate methods, RT-PCR, single-cell RT-PCR, and in situ RT-PCR, were used to analyze clonotypic cells in peripheral blood mononuclear cells (PBMC) from MM patients. Sequencing of the IgH transcripts expressed by individual cells obtained by limiting dilution of freshly isolated PBMC from a MM patient showed that all B cells expressed an identical CDR3. This intraclonal homogeneity indicates an escape from antigenic-selection, characteristic of malignant B cells. For this patient, the frequency of clonotypic PBMC, about 25%, was comparable to the number of PBMC B cells (34%). Because the PBMC included less than 1% plasma cells, virtually all clonotypic PBMC must be B cells. Using single-cell RT-PCR, clonotypic IgH transcripts were identified in individual sorted B cells from blood. To accurately quantify the number of clonotypic B cells, sorted B cells derived from 18 MM patients (36 samples) and 18 healthy donors (53 samples) were analyzed using in situ RT-PCR with patient-specific primers. Clonotypic transcripts were not detectable among normal B cells. For the 18 MM patients, a mean of 66% +/- 4% (SE) of blood B cells were clonotypic (range, 9% to 95%), with mean absolute number of 0.15 +/- .02 x 10(9)/L blood. Over time in individual patients, conventional chemotherapy transiently decreased circulating clonotypic B cells. Their numbers were increased in granulocyte colony-stimulating factor (G-CSF)- mobilized blood of one patient. However, clonotypic B cells of a one patient became undetectable after allogeneic transplant, correlating with complete remission. Although contributions to MM spread and progression is likely, their malignant status and impact has yet to be clarified. Their high frequency in the blood, and their resistence to conventional chemotherapy suggests that the number of circulating clonotypic cells should be clinically monitored, and that therapeutic targeting of these B cells may benefit myeloma patients.

Lymph node modification in patients with the acquired immunodeficiency syndrome (AIDS) or with AIDS related complex (ARC). A histological, immuno-histopathological and ultrastructural study of 45 cases.

The authors present the results of a histopathological study on the lymph-nodes taken from 45 subjects suffering from either an AIDS or from a chronic adenopathy corresponding to the definition of AIDS related complex (ARC). The various aspects observed were classed as type I to type IV. The lymph-node modifications observed in the 29 patients with an ARC could be divided into three principle groups: an extensive follicular hyperplasia associated with other elementary lesions or type IA (25 lymph-nodes from 23 patients); changes resembling a multicentric Castleman syndrome or type IB (1 case); angioimmunoblastic-like (AIL) lesions or type II (2 cases) and an association of lesions of type II (7 lymph-nodes from 6 patients). During AIDS, the adenopathy usually disappears, and the small lymph-nodes removed, especially on autopsy, show an extensive lymphoid depletion (type III) with systematic sclerosis (15 lymph-nodes from 14 patients). When adenopathy persists, it is due to infections complications (tuberculosis, cryptococcosis, avian mycobacteriosis and Whipple's disease like lesions). Of the 10 patients in whom a Kaposi's sarcoma was observed, only 6 showed lymph-node involvement, or type IV. The different histopathological lesions seem to appear according to an evolving succession, proven by certain association of lesions and by successive biopsies. In our series, 17% of subjects with an ARC evolved to AIDS. Lymph-node biopsy allows a possible ARC to be implicated on the association of the following simple lesions: follicular hyperplasia with partial or total destruction of the perifollicular lymphocytic cisterna, infiltration of the germinative centres by streams of small lymphocytes, evolving to an aspect of a "burst" germinative centre and various sinusal reactions with, in particular, the presence of neutrophilic polynuclear cells. The biopsy also allows the forms with bad prognosis to be recognized: those with AIL-like aspect or multicentric Castleman-like syndrome, which seems to represent a particular evolutive form. Finally, it also detects, in certain cases, the localization of a Kaposi syndrome, signalling the passage to AIDS. The immunopathological studies present a double interest. Firstly, they offer arguments in favour of the diagnosis: increase in the number of T8 lymphocytes in the germinative centres with the formation of small clusters and disruption of the network of dendritic reticular cells, and the inversion of the T4/T8 ratio in the extra-follicular cortical regions, by either a decrease in T4 lymphocytes or by an increase in T8 lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)