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Inicio Medicina Clínica CD64, CD11a and CD18 leukocytes expression in children with SARS-CoV-2 multisyst...
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Vol. 156. Núm. 2.
Páginas 89-91 (Enero 2021)
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Vol. 156. Núm. 2.
Páginas 89-91 (Enero 2021)
Scientific letter
DOI: 10.1016/j.medcli.2020.09.002
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CD64, CD11a and CD18 leukocytes expression in children with SARS-CoV-2 multisystem inflammatory syndrome versus children with Kawasaki disease: Similar but not the same
Comparación de la expresión de CD64, CD11a y CD18 en leucocitos de niños con síndrome inflamatorio multisistémico relacionado con SARS-CoV-2 y enfermedad de Kawasaki: semejantes pero distintos
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Alberto García-Salidoa,
Autor para correspondencia
citopensis@yahoo.es

Corresponding author.
, Sandra Cuenca-Carceléna, Ana Castillo-Robledab
a Pediatric Critical Care Unit, Hospital Infantil Universitario Niño Jesús, Madrid, Spain
b Pediatric Oncohematology Unit, Flow Cytometry Laboratory, Hospital Infantil Universitario Niño Jesús, Madrid, Spain
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Table 1. Clinical trajectories of Kawasaki disease and SARS-COV2 infected children and cell expression of CD64, CD18, and CD11a.
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Dear Editor,

The immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to be a critical factor in the prognosis of coronavirus disease 2019 (COVID-19).1 Generally, children are less affected and developed asymptomatic or mild forms. Despite this, pediatricians across Europe have describe severe cases of the disease. Firstly recognized as “Kawasakilike” processes and later named as pediatric multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS).2–4 We have seen similar cases in our center adding to its clinical and analytical study the application of immunophenotyping by flow cytometry (FC).4

In this report, we describe CD64, CD18, and CD11a expression in three children with PIMS-TS and compare it with three cases of Kawasaki Disease (KD, years 2018 and 2019). The CD64 is a type I high-affinity receptor for the Fc fraction of the immunoglobulin G, located on the surface of monocytes, macrophages, dendritic cells, and neutrophils. Increased CD64 on the cell surface is related to the intensity of stimulation received by inflammatory cytokines. Additionally, CD18, also known as integrin β2, participates in leukocyte adhesion and signaling. CD11a associates with CD18 to form the lymphocyte function-associated antigen 1, or LFA-1. Expressed on leukocytes, this T cell integrin plays a central role in leukocyte cell-cell interactions and lymphocyte stimulation.

The cases clinical trajectories are described in Table 1. The children were studied after informed consent obtained from their parents or legal guardians. The samples were collected in sterile EDTA at room temperature, refrigerated at 4°C and analyzed by FC within 24h. Cell surface expression of CD64, CD18, and CD11a were measured by BD FACS Canto II flow cytometer (Becton Dickinson, New York, USA). CD64 (clone 10.1), CD18 (clone CBR LFA-1/2), and CD11a (clone HI111) monoclonal antibodies were obtained from Biolegend® (San Diego, CA, USA). Expressions were measured in monocytes, neutrophils and lymphocytes were identified on a dot-plot and gated. Cell viability was confirmed by 7-AAD staining. At least 10,000 events were recorded for each sample. Flow-cytometry settings and samples were prepared according to manufacturer instructions. The intensity of CD64, CD18, and CD11a surface expression were measured as mean fluorescence intensity in arbitrary units (MFI).

Table 1.

Clinical trajectories of Kawasaki disease and SARS-COV2 infected children and cell expression of CD64, CD18, and CD11a.

Clinical trajectories of Kawasaki disease and SARS-COV2 infected children
Patient  Age in years  Sex  Days of fever  KD Clinical features at admission  KD Laboratory criteria  Echocardiogram  Treatment  Response 
KD1  Female  C, E, X, O  1, 2, 7, 8  Normal heart function  Ig 1d  No fever from beginning 
KD2  Female  C, E, X, O  1, 2, 6  Normal heart function  Ig 1d  Low grade fever less than 24
KD3  Female  C, E, X, O  1, 2, 6, 8  Normal heart function  Ig 2d  Persistent fever after 48h, requiring a second dose of Ig 
SARS-COV2 1(IgG antibodies to SARS-CoV-2)  Male  1, 6, 7  Normal heart function  Methylprednisolone(1mg/kg/day)  Recovered, 10 days of PICU admission 
SARS-COV2 2(Confirmed by RT-PCR from nasopharyngeal swab)  11  Female  X, C  1, 6, 7  Depressed cardiac function  Methylprednisolone(1mg/kg/day), Ig 1d, inotropic support  Recovered, 8 days of PICU admission 
SARS-COV2 3(Confirmed by RT-PCR from nasopharyngeal swab)  11  Male  X, C  1, 3, 6, 7  Normal heart function  Methylprednisolone(1mg/kg/day), tocilizumab, inotropic support  Recovered, 4 days of PICU admission 
Cell surface expression as mean fluorescence intensity in arbitrary units of CD64, CD18, and CD11a.
  % Neutrophils CD64+  Neutrophils CD64  Neutrophils CD11a  Neutrophils CD18  Monocytes CD64  Monocytes CD11a  Monocytes CD18  Lymphocites CD8/CD11a  Lymphocites CD8/CD18 
KD1  78.1  3070  2354  5918  12049  6101  9136  2731  4821 
KD2  78.7  2045  3323  16821  11222  12,721  17812  5313  6545 
KD3  83.5  2315  3304  9529  9761  9411  15175  6608  11919 
SARS-COV2 1  99.6  14526  6123  3010  34065  18564  4762  21648  1703 
SARS-COV2 2  99.8  20940  9315  11679  51403  35081  13466  20414  1617 
SARS-COV2 3  99.6  17218  6458  6134  45095  19484  8695  33722  2124 

KD: Kawasaki disease.

KD Clinical features: E: changes in limbs; X: polymorphous exanthema; C: bilateral conjunctival injection; O: changes in oral cavity or lips; L: unilateral cervical lymphadenopathy>1.5cm.

KD Laboratory criteria: 1=CRP>3mg/dl, 2=ESR>40mm/h, 3=WBC count>15,000/mm3; 4=normocytic anemia for age; 5=platelets after fever for 7 d>450,000/mm3; 6=ALT>45IU/L; 7=albumin<3.0g/dL; and 8=urine>10 WBCs/high-power field.

Legend and experiment data: The children were studied after informed consent obtained from their parents or legal guardians. The samples obtained were collected in sterile EDTA at room temperature or refrigerated at 4°C and analyzed by FC within 24h. Cell surface expression of CD64, CD18, and CD11a was measured by BD FACS Canto II flow cytometer (Becton Dickinson, New York, USA). CD64 (clone 10.1), CD18 (clone CBR LFA-1/2), and CD11a (clone HI111) monoclonal antibodies were obtained from Biolegend® (San Diego, CA, USA). Expressions were measured in monocytes, neutrophils, and lymphocytes. Cell viability was confirmed by 7-AAD staining. At least 10,000 events were recorded for each sample. Flow-cytometry settings and samples were prepared according to manufacturer instructions. Neutrophils, monocytes, and lymphocytes were identified on a dot-plot and gated. The intensity of CD64, CD18, and CD11a surface expression were measured as mean fluorescence intensity in arbitrary units (MFI).

The expression of CD64, CD11a, and CD18 are in Table 1. All PIMS-TS cases received methylprednisolone prior to FC, the KD cases were studied before received immunoglobulin. As main finding, we observe higher upregulation of the three proteins studied in SARS-CoV-2 patients. This response appears to be similar but higher than in KD.

Recent papers have described that a dysregulated immune response may result in inflammation and clinical worsening in COVID-19 cases. Our cases show high levels of CD64 expression. This expression is also higher than the described in some autoinflammatory diseases, so it could indicate immune dysregulation. Cytokines such as IL-1b, IL-6, and TNF-a affect the presence of CD64 on the cell surface.5

Regarding the LFA-1, it is known that plays a key role in leukocyte migration into tissues. Lymphopenia is a common finding in COVID-19 patients.1 This situation may be linked to the migration of CD8+ lymphocytes to the infected tissues. As seen in Table 1, the CD11a upregulation in CD8+ is clear and could be linked to this process. Additionally, LFA-1 is involved in the process of cytotoxic T cell-mediated killing as well as antibody-mediated killing by granulocytes and monocytes. We observed an exacerbated CD8+ LFA-1 expression compared to KD cases.

The nonantimicrobial COVID-19 therapies proposed are intended to downregulate the immune system. The cytokine storm theory, coupled with analytical data that suggest immune dysregulation or macrophage activation syndrome justify these therapies. In our cases, all FC analyses were performed after almost four days following disease onset. Also, in one case FC was conducted in a patient with positive immunoglobulin G to SARS-CoV-2, which is not a precocious immune reaction.1 The observation of high CD64 expression helped to detect a proinflammatory status and helped to take the decision of using immunoregulatory therapies.4

In summary, we compare for the first time the immunophenotype of children with severe SARS-CoV-2 infection versus children with KD. We observed significant but higher upregulation of CD64, CD18, and CD11a expression on leukocytes. Prospective studies with a higher number of cases should be conducted to confirm this observation.

Funding

This study was partially funded by “Fundación Familia Alonso”.

Conflict of interest

No conflict interest.

References
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C. Qin, L. Zhou, Z. Hu, S. Zhang, S. Yang, Y. Tao, et al.
Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, China.
Clin Infect Dis, 71 (2020), pp. 762-768
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An outbreak of severe Kawasaki-like disease at the Italian epicentre of the SARS-CoV-2 epidemic: an observational cohort study.
Lancet, 395 (2020), pp. 1771-1778
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Severe SARS-CoV-2 infection in children with suspected acute abdomen: a case series from a tertiary hospital in Spain.
Pediatr Infect Dis J, 39 (2020), pp. e195-e198
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T. Yamazaki, S. Hokibara, T. Shigemura, N. Kobayashi, K. Honda, Y. Umeda, et al.
Markedly elevated CD64 expressions on neutrophils and monocytes are useful for diagnosis of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome during flares.
Clin Rheumatol, 33 (2014), pp. 677-683
Copyright © 2020. Elsevier España, S.L.U.. All rights reserved
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